Microbiota restoration therapy (mrt) compositions and methods of manufacture

ABSTRACT

Microbiota restoration therapy (MRT) compositions (e.g., oral MRT compositions) and methods for manufacturing MRT compositions are disclosed. An example method for manufacturing an MRT composition may include collecting a stool sample, purifying the stool sample to form a purified sample, stabilizing the purified sample to form a stabilized sample, converting the stabilized sample to a solid, adding one or more additives and/or excipients to the solid to form a treatment composition, and encapsulating the treatment composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of pending U.S. applicationSer. No. 15/178,176, filed Jun. 9, 2016, which claims priority under 35U.S.C. § 119 to U.S. Provisional Application Ser. No. 62/173,182, filedJun. 9, 2015 and U.S. Provisional Application Ser. No. 62/247,825, filedOct. 29, 2015, the entirety of which are incorporated herein byreference.

FIELD

The present disclosure pertains to compositions and methods for treatingpatients.

BACKGROUND

A wide variety of compositions and methods have been developed fortreating diseases and/or conditions of the digestive track. Of the knowncompositions and methods, each has certain advantages and disadvantages.There is an ongoing need to provide alternative compositions and methodsfor treating diseases and/or conditions of the digestive track.

BRIEF SUMMARY

This disclosure provides design, material, manufacturing method, and usealternatives for compositions and methods for treating patients. Anexample method for manufacturing an oral microbiota restoration therapy(MRT) composition is disclosed. The method comprises:

collecting a stool sample;

purifying the stool sample to form a purified sample;

stabilizing the purified sample to form a stabilized sample;

converting the stabilized sample to a solid;

adding one or more additives and/or excipients to the solid to form atreatment composition; and

encapsulating the treatment composition.

An example method for manufacturing an oral microbiota restorationtherapy (MRT) composition is disclosed. The method comprises:

collecting a stool sample;

purifying the stool sample to form a purified intermediate, whereinpurifying the stool sample comprises:

-   -   adding a diluent to the stool sample;    -   mixing the stool sample and diluent to form a mixture;    -   filtering the mixture;    -   transferring a filtrate from the filtering step to a centrifuge        tube; and    -   centrifuging the filtrate to arrive at the purified        intermediate;

lyophilizing the purified intermediate to form a plurality lyophilizedpellets; and

encapsulating the plurality of lyophilized pellets in one or morecapsules.

Alternatively or additionally to any of the embodiments above, filteringthe mixture comprises filtering the mixture to obtain a sample havingparticles in the range of 50 to 70 micrometers (μm).

Alternatively or additionally to any of the embodiments above,centrifuging the filtrate comprises centrifuging the filtrate at a ratesuch that the centrifugal force is in the range of about 8-12,000 g forin the range of 15 to 45 minutes.

Alternatively or additionally to any of the embodiments above,lyophilizing the purified intermediate comprises the steps of:

mixing the purified intermediate with a lyophilization excipient to forma lyophilization intermediate;

placing the lyophilization intermediate into a plate having a pluralityof wells; lowering a temperature of the lyophilization intermediate to atemperature in the range of −40 to −45° C.;

applying a vacuum to the lyophilization intermediate and raising thetemperature of the lyophilization intermediate to approximately 0° C.;

initializing a secondary drying step and raising the temperature of thelyophilization intermediate to approximately 25° C.;

releasing the vacuum; and

removing a plurality of lyophilized pellets from the plate.

Alternatively or additionally to any of the embodiments above, thelyophilization excipient comprises at least 2.3% PEG 3350, 1% glycerin,10% trehalose, and 10% sucrose.

Alternatively or additionally to any of the embodiments above, the oneor more capsules comprise hypromellose capsule.

Alternatively or additionally to any of the embodiments above, furthercomprising banding the capsules.

Alternatively or additionally to any of the embodiments above, thebanding material comprises hypromellose, an anionic copolymer based onmethacrylic acid and methyl methacrylate, hypromellose phthalate, orhypromellose acetate succinate.

A method for manufacturing an oral microbiota restoration therapy (MRT)composition is disclosed. The method comprises:

adding a diluent to a purified stool sample, the purified stool samplecomprising stool and a solution of 2.3% cryoprotectant and 0.9% sodiumchloride solution;

mixing the stool sample and diluent to form a mixture;

filtering the mixture;

transferring a filtrate from the filtering step to a centrifuge tube;and

centrifuging the filtrate to arrive at the purified intermediate;

lyophilizing the purified intermediate to form a plurality lyophilizedpellets; and

encapsulating the plurality of lyophilized pellets in one or morecapsules.

Alternatively or additionally to any of the embodiments above, filteringthe mixture comprises filtering the mixture to obtain a sample havingparticles in the range of 50 to 70 micrometers (μm).

Alternatively or additionally to any of the embodiments above,centrifuging the filtrate comprises centrifuging the filtrate at a ratesuch that the centrifugal force is in the range of about 8-12,000 g forin the range of 15 to 45 minutes.

Alternatively or additionally to any of the embodiments above,lyophilizing the purified intermediate comprises the steps of:

mixing the purified intermediate with a lyophilization excipient to forma lyophilization intermediate;

placing the lyophilization intermediate into a plate having a pluralityof wells;

lowering a temperature of the lyophilization intermediate to atemperature in the range of −40 to −45° C.;

applying a vacuum to the lyophilization intermediate and raising thetemperature of the lyophilization intermediate to approximately 0° C.;initializing a secondary drying step and raising the temperature of thelyophilization intermediate to approximately 25° C.;

releasing the vacuum; and

removing a plurality of lyophilized pellets from the plate.

Alternatively or additionally to any of the embodiments above, thelyophilization excipient comprises at least 2.3% PEG 3350, 1% glycerin,10% trehalose, and 10% sucrose.

Alternatively or additionally to any of the embodiments above, the oneor more capsules comprise hypromellose capsule.

Alternatively or additionally to any of the embodiments above, furthercomprising banding the capsules.

Alternatively or additionally to any of the embodiments above, thebanding material comprises hypromellose, an anionic copolymer based onmethacrylic acid and methyl methacrylate, hypromellose phthalate, orhypromellose acetate succinate.

Alternatively or additionally to any of the embodiments above, furthercomprising packaging the encapsulated lyophilized pellets into packetsin individual dosage quantities.

Alternatively or additionally to any of the embodiments above, thepackets comprises metallized polyester/polyethylene bonded film.

Alternatively or additionally to any of the embodiments above, furthercomprising placing the packets into one or more child-resistantcontainers.

Alternatively or additionally to any of the embodiments above, furthercomprising packaging the encapsulated lyophilized pellets into packetsin individual dosage quantities.

Alternatively or additionally to any of the embodiments above, thepackets comprises metallized polyester/polyethylene bonded film.

Alternatively or additionally to any of the embodiments above, furthercomprising placing the packets into one or more child-resistantcontainers.

The above summary of some embodiments is not intended to describe eachdisclosed embodiment or every implementation of the present disclosure.The Figures and Detailed Description which follow more particularlyexemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure may be more completely understood in consideration of thefollowing detailed description of various embodiments of the disclosurein connection with the accompanying drawings, in which:

FIG. 1 is a flowchart depicting an overall process for manufacturing astandardized FMT composition; and,

FIG. 2 is a flowchart depicting further steps in a representativemanufacturing process.

FIG. 3 is a flowchart depicting further steps in another representativemanufacturing process.

FIG. 4 is a flowchart depicting further steps in another representativemanufacturing process.

FIG. 5 is a flowchart depicting further steps in another representativemanufacturing process.

While the disclosure is amenable to various modifications andalternative forms, specifics thereof have been shown by way of examplein the drawings and will be described in detail. It should beunderstood, however, that the intention is not to limit the disclosureto the particular embodiments described. On the contrary, the intentionis to cover all modifications, equivalents, and alternatives fallingwithin the spirit and scope of the disclosure.

DETAILED DESCRIPTION

For the following defined terms, these definitions shall be applied,unless a different definition is given in the claims or elsewhere inthis specification.

All numeric values are herein assumed to be modified by the term“about,” whether or not explicitly indicated. The term “about” generallyrefers to a range of numbers that one of skill in the art would considerequivalent to the recited value (i.e., having the same function orresult). In many instances, the terms “about” may include numbers thatare rounded to the nearest significant figure.

The recitation of numerical ranges by endpoints includes all numberswithin that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4,and 5).

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contentclearly dictates otherwise. As used in this specification and theappended claims, the term “or” is generally employed in its senseincluding “and/or” unless the content clearly dictates otherwise.

It is noted that references in the specification to “an embodiment”,“some embodiments”, “other embodiments”, etc., indicate that theembodiment described may include one or more particular features,structures, and/or characteristics. However, such recitations do notnecessarily mean that all embodiments include the particular features,structures, and/or characteristics. Additionally, when particularfeatures, structures, and/or characteristics are described in connectionwith one embodiment, it should be understood that such features,structures, and/or characteristics may also be used connection withother embodiments whether or not explicitly described unless clearlystated to the contrary

The following detailed description should be read with reference to thedrawings in which similar elements in different drawings are numberedthe same. The drawings, which are not necessarily to scale, depictillustrative embodiments and are not intended to limit the scope of thedisclosure.

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees, and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be included within the scope of this term.

The term “cryopreservation,” as used herein, refers to the process ofcooling and storing biological cells, tissues, or organs at very lowtemperatures to maintain Their viability. As a example, cryopreservationcan be the technology of cooling and storing cells at a temperaturebelow the freezing point (e.g., 196 K) that permits high rates ofsurvivability of the cells upon thawing.

The term “cryoprotectant,” as used herein, refers to a substance that isused to protect biological cells or tissues from the effects offreezing.

As used herein, the term “microbiota” can refer to the human microbiome,the human microbiota or the human gut microbiota. The human microbiome(or human microbiota) is the aggregate of microorganisms that reside onthe surface and in deep layers of skin, in the saliva and oral mucosa,in the conjunctiva, and in the gastrointestinal, genito-urinary, orvaginal tracts of humans. The human microbiome is comprised of bacteria,fungi, and archaea. Some of these organisms perform tasks that areuseful for the human host, but the function of the majority of theorganisms that make up the human microbiome is unknown. Under normalcircumstances, these microorganisms do not cause disease to the humanhost, but instead participate in maintaining health. Hence, thispopulation of organisms is frequently referred to as “normal flora.”

The population of microorganisms living in the human gastrointestinaltract is commonly referred to as “gut flora” or “gut microbiota.” Themicrobial flora of the human gut encompasses a wide variety ofmicroorganisms that aid in digestion, the synthesis of vitamins, andcreating enzymes not produced by the human body.

The phrase “microbiota restoration therapy,” as used herein, refers to acomposition which may include, but is not limited to, human fecalmaterial containing viable gut flora from a patient or donor, a diluent,and a cryoprotectant. Additional compositions include equivalentfreeze-dried and reconstituted feces or a “synthetic” fecal composition.The human fecal material is screened for the presence of pathogenicmicroorganisms prior to its use in the microbiota restoration therapy.The human fecal material is screened for the presence of Clostridiumspecies including C. difficile, Norovirus, Adenovirus, entericpathogens, antigens to Giardia species, Cryptosporidia species and otherpathogens, including acid-fast bacteria, enterococci, including but notlimited to vancomycin-resistant enterococci (VRE), methicillin-resistantStaphylococcus aureus (MSRA), as well as any ova or parasitic bodies, orspore-forming parasites, including but not limited to Isospora,Clyslospora, and Cryptospora.

The process of fecal bacteriotherapy can include introducing a fecalsample of a healthy donor, or a donor having one or more desiredcharacteristics, into a gastrointestinal tract of a patient torepopulate a healthy or desirable gut microbiota. In certain examples,prior to introduction of the fecal sample, the patient's intestinalflora can be disrupted using antibiotics, such that the healthy ordesirable gut microbiota, once introduced into the patient, can easilypopulate the gastrointestinal tract.

The human fecal material is optionally filtered prior to its use in themicrobiota restoration therapy.

The present disclosure is directed to compositions, methods ofmanufacture and methods of treatment utilizing microbiota restorationtherapy (MRT) for the treatment of Clostridium difficile infections(CDI). CDI is a common nosocomial infection and is frequently associatedwith severe morbidity and mortality, especially in elderly patients.While CDI treatment is one example use for the MRT compositionsdisclosed herein, this is not intended to be limiting. Other diseasesand/or conditions are contemplated. Some of the medical conditions thatmay be desirably impacted by treatment with MRT compositions may includecardiovascular and/or peripheral vascular disease, allergies, obesity,hypoglycemia, constipation, celiac sprue (e.g., celiac disease),gastrointestinal cancer (e.g. gastrointestinal cancer is at least one ofstomach cancer, esophageal cancer, colon cancer gallbladder cancer,liver cancer, pancreatic cancer, colorectal cancer, anal cancer, andgastrointestinal stromal tumors), melanoma, non-squamous cell lungcancer, squamous cell lung cancer, renal cell carcinoma, head and necktumors, bladder cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, gastriccancer, colorectal cancer, multiple myeloma, esophageal cancer, breastcancer, glioblastoma, mediastinal B-cell lymphoma, other hematologicmalignancies, testicular cancer, pancreatic cancer, lymphoma, cervicalcancer, ovarian cancer, basal cell carcinoma, neuroblastoma, leukemia,sarcoma, other cancers, myoclonus dystonia, sacrolileitis,spondyloarthropatliy, spondylarthritis, proximal myotonic myopathy; anautoimmune disease nephritis syndrome, autism, travelers' diarrhea,small intestinal bacterial overgrowth, chronic pancreatitis, apancreatic insufficiency, chronic fatigue syndrome, benign myalgicencephalomyelitis, chronic fatigue immune dysfunction syndrome,Parkinson's Disease (PD), amyotrophic lateral sclerosis (ALS), multiplesclerosis (MS), degenerative neurological diseases, Grand mal seizuresor petitmal seizures, Steinert's disease, chronic infectiousmononucleosis, epidemic myalgic encephalomyelitis, idiopathicthrombocytopenic purpura (ITP), an acute or chronic allergic reactionobesity, anorexia, irritable bowel syndrome (IBS or spastic colon)Crohn's disease, irritable bowel disease (IBD), colitis, ulcerativecolitis or Crohn's colitis, chronic infectious mononucleosis, epidemicmyalgic encephalomyelitis, acute or chronic urticarial, lupus,rheumatoid arthritis (RA) or juvenile idiopathic arthritis (JIA),pre-diabetic syndrome, fibromyalgia (FM), Type I or Type II diabetes,acute or chronic insomnia, migraines, and attentiondeficit/hyperactivity disorder (ADHD).

In the case of humans, the present disclosure encompasses methods oftreatment of chronic disorders associated with the presence of abnormalenteric microflora. Such disorders include but are not limited to thoseconditions in the following categories: gastro-intestinal disordersincluding irritable bowel syndrome or spastic colon, functional boweldisease (FBD), including constipation predominant FBD, pain predominantFBD, upper abdominal FBD, nonulcer dyspepsia (NUD), gastro-oesophagealreflux, inflammatory bowel disease including Crohn' s disease,ulcerative colitis, indeterminate colitis, collagenous colitis,microscopic colitis, chronic Clostridium difficile infection,pseudemembranous colitis, mucous colitis, antibiotic associated colitis,idiopathic or simple constipation, diverticular disease, AIDSenteropathy, small bowel bacterial overgrowth, coeliac disease,polyposis coil, colonic polyps, chronic idiopathic pseudo obstructivesyndrome; chronic gut infections with specific pathogens includingbacteria, viruses, fungi and protozoa; viral gastrointestinal disorders,including viral gastroenteritis, Norwalk viral gastroenteritis,rotavirus gastroenteritis, AIDS related gastroenteritis; liver disorderssuch as primary biliary cirrhosis, primary sclerosing cholangitis, fattyliver or cryptogenic cirrhosis; rheumatic disorders such as rheumatoidarthritis, non-rheumatoid arthritidies, non rheumatoid factor positivearthritis, ankylosing spondylitis, Lyme disease, and Reiter' s syndrome;immune mediated disorders such as glomeruionephritis, haemolytic uraemicsyndrome, juvenile diabetes mellitus, mixed cryoglobulinaemia,polyarteritis, familial Mediterranean fever, amyloidosis, scleroderma,systemic lupus erythematosus, and Behcets syndrome; autoimmune disordersincluding systemic lupus, psoriasis, idiopathic thrombocytopenicpurpura, Sjogren's syndrome, haemolytic uremic syndrome or scleroderma;neurological syndromes such as chronic fatigue syndrome, migraine,multiple sclerosis, amyotrophic lateral sclerosis, myasthenia gravis,Guillain-Barre syndrome, Parkinson's disease, Alzheimer's disease,Chronic Inflammatory Demyelinating Polyneuropathy, and otherdegenerative disorders; psychiatric disorders including chronicdepression, schizophrenia, psychotic disorders, manic depressiveillness; regressive disorders including, Asbergers syndrome, Rettsyndrome, attention deficit hyperactivity disorder (ADHD), and attentiondeficit disorder (ADD); the regressive disorder, autism; sudden infantdeath syndrome (SIDS), anorexia nervosa; dermatological conditions suchas chronic urticaria, acne, dermatitis herpetiformis and vasculitisdisorders; and cardiovascular and/or vascular disorders and diseases.

Globally, the increase in the prevalence of drug resistant organisms hascreated many challenges for clinicians that may pose public healthrisks. Infections by drug resistant organisms (e.g.,vancomycin-resistant Enterococcus (VRE)) and Clostridium difficileinfection share similar risk factors. VRE is a nosocomial pathogen thatcan be a complication among transplant and immune compromised patients.VRE carriers may also be at increased risk for infection due to VRE andalso be a potential source of VRE transmissions to others. VRE sheddingin stool increases with antimicrobial exposures and decreases withnormalization of the intestinal microbiota after antimicrobials arediscontinued. Accordingly, normalization of intestinal microbiota maynot only be useful for treating Clostridium difficile infections(including chronic infections), these treatments may also be useful fortreating infections by drug resistant organisms (e.g., VRE and/or otherdrug resistant organisms including those disclosed herein).

In some instances, the microbiota restoration therapy compositions(and/or fecal bacteriotherapy compositions) disclosed herein may be usedto treat patients with infections by drug resistant organisms and/ormulti-drug resistant organisms (MDRO). The drug resistant organisms maybe resistant to antimicrobial agents (e.g., antibiotics, antivirals,antifungals, antiparasitics, other drugs, combinations thereof, and thelike) and may include drug resistant micro-organisms such as bacteria,viruses, fungi, parasites, etc. The infections that can be treated bythe microbiota restoration therapy compositions disclosed herein may bealong the digestive tract or along other systems of the patient.

The microbiota restoration therapy compositions may be used to treatinfections by a variety of drug resistant organisms such asvancomycin-resistant enterococci (VRE), methicillin-resistantStaphylococcus aureus (MRSA), extended-spectrum β-lactamase producinggram-negative bacteria, Klebsiella pneumoniae carbapenemase producinggram-negative bacteria, multi-drug resistant gram negative rods bacteria(e.g., such as Enterobacter species, E. coli, Klebsiella pneumoniae,Acinetobacter baumannii, and Pseudomonas aeruginosa), drug resistantEnterobacter species, multi-drug resistant tuberculosis (e.g.,Mycobacterium tuberculosis), drug resistant staphylococci, drugresistant enterococci, drug resistant gonococci, drug resistantstreptococci (e.g., including Streptococcus pneumoniae), drug resistantsalmonella, drug resistant gram negative bacteria, drug resistantCandida, drug resistant HIV, drug resistant influenza virus, drugresistant cytomegalovirus, drug resistant herpes simplex virus, drugresistant malaria, drug resistant Plasmodium vivax, drug resistantPlasmodium falciparum, drug resistant Toxoplasma gondii, and the like,and/or other drug resistant organisms. These are just examples.

The microbiota restoration therapy compositions may be used to treatother infections including urinary tract infections.

Treatment of infections by drug resistant organisms with the microbiotarestoration therapy compositions disclosed herein may include treatingpatients with no prior history of infection with a drug resistantorganism, treating patients with a single prior infection by a drugresistant organism, treating patients with two or more (e.g., two,three, four, five, six, or more) prior infections by a drug resistantorganism, etc. In some instances, the microbiota restoration therapycompositions may be used to treat a patient with three prior infectionsby a drug resistant organism. In other instances, the microbiotarestoration therapy compositions may be used to treat a patient with twoprior infections by a drug resistant organism if the prior infectionsresulted in hospitalization, if the prior or current infections requiretreatment with toxic drugs, or if the prior infections were all from thesame organism.

In some instances, MRT compositions can be administered to a patientusing an enema or other suitable technique. However, it may be desirableto orally administer an MRT composition. In order to prepare an MRTcomposition in a form suitable for oral administration, a number ofsteps may be carried out. Generally, these steps may include collectinga fecal sample, processing the fecal sample, lyophilizing or“freeze-drying” the processed fecal sample (or otherwise converting theprocessed fecal sample from a liquid to a solid), adding one or moreadditives and/or excipients, and forming an oral form of the MRTcomposition from the lyophilized material and additives (e.g., a tablet,capsule, liquid preparation, or the like). Some additional detailsregarding at least some of these steps are disclosed herein.

FIG. 1 is a flow chart depicting a portion of an example MRT productionprocess. This is just an example. Other examples of screening donors,obtaining human stool samples, and processing the stool samples to a MRTproduct are disclosed in commonly assigned U.S. Patent Publication2014/0363398, which is herein incorporated by reference. Moreparticularly, FIG. 1 schematically depicts a process for collecting andinspecting a donor fecal sample. As a first step in thecollecting/inspecting process, potential stool donors are screened.Screening/prescreening is described in more detail herein. Once thedonor passes the screening, step two may include collecting the donor'sstool using a human stool collection kit as defined herein, whether athome or at a collection facility. The kit can include, but is notlimited to, a clean human stool collection container with lid, a largecloseable/sealable bag, a donation form and a human stool collectioninstruction sheet. The time and date of collection, along with donoridentity and method of transport, can be recorded in order to track thetime from collection to processing, and the conditions of transport. Asa non-limiting example, the collection container can include anindicator of the minimum and the maximum temperature to which the sampleis exposed. As another non-limiting example, one or more temperaturesensitive stickers that changes color at temperatures below about 4° C.and temperatures greater than about room temperature (about 22-29° C.)can be affixed to the container.

Step three may involve transporting the sample to a processing facility.It can be appreciated that if the sample is collected at the processingfacility, transporting the sample is not necessary. In some instances itmay be desirable to collect the sample at the processing facility inorder to more clearly establish the chain of custody of the sample. Withthe receipt of the first stool donation for any individual, a profilewill be established for each donor. Subsequent stool samples can besubjected to a human stool test, which is utilized to match and confirmthe identity of the donor with the donation. Based on prior collectedsamples, a human stool profile for the donor is generated and can bemaintained or enhanced over repeated donations. Any new sample will becompared with this profile to confirm it is the same donor.Differentiation can be made to confirm donor identity based on therepresentation of Bacterioides species in the human stool. In anon-limiting example, the base set of stool samples used to create theprofile is collected at the processing facility to assure donor identityin the profile samples. In another non-limiting example, the base set ofstool samples used to create the profile can be collected in locationsother than the processing facility, with donor identity assuranceprotocols appropriate to the situation or location.

Step four of the method may include labeling the donation “Quarantine”and holding the donation in quarantine at or below room temperature forno longer than in the range of 24 hours to five days prior toprocessing. Donations may be rejected in situations where thetemperature indicator has been activated or where the time betweendonation and receipt exceeds 24 hours. In addition, where applicable,the human stool test results must match the donor profile. If the humanstool test does not match the donor profile, the donation collected forthat day will be discarded and the donor will be disqualified.

In one method of the disclosure, the human stool sample is processedwithin about 24 hours of collection. In another method of theapplication, the time of collection is recorded at the time of arrivalof the stool sample at the processing facility. Step six may includeinspecting the stool donation. Visual inspection can be completed uponarrival of the stool sample at the processing facility. In the event thehuman stool sample is loose, unformed, is not of sufficient weight(e.g., less than about 50 g), or for any other reason, including but notlimited to evidence indicating poor sample quality or concerns aboutdonor health, the sample may be rejected, labeled “Inspection-Rejected”and the donation is discarded. Further, answers to questions on thehuman stool collection form can be reviewed by trained personnel.Certain answers in the collection form may require ample rejection. Ifthe sample is accepted, it may be labeled

“Inspection-Accepted” and may be moved to a manufacturing process.

FIG. 2 is a flow chart depicting a portion of a generic illustrativemethod for preparing a stool sample for MRT as an oral dosage. It iscontemplated that an intermediate product within the method forpreparing a stool sample for MRT as an oral dosage may be suitable forMRT via an enema or gastro-nasal tube. The stool sample may first becollected and screened 100, for example, in the method described withrespect to FIG. 1. Once the sample has been accepted, the sample may bepurified and concentrated 102. The sample may be purified usingcentrifugation, membrane filtration, or a combination thereof to removefecal material above a certain particle size. It is contemplated thatsince most bacteria of interest are in the range of 0.3 microns (μm) to30 μm, the sample may be processed to remove particles greater than50-70 μm. The sample may be processed to obtain a 75% to 90%concentration of the bacteria. This may allow for an increasedflexibility in the ratio of formulation excipients to bacteria forfurther processing.

The sample may be membrane filtered in a number of different ways,including, but not limited to the use of filter bags, pressure filters,and/or vacuum filters. In some instances, the sample may be filteredmultiple times using a smaller filter membrane with each subsequentfiltering. In some instances, saline may be added as a diluent in aratio of 1:3 (stool to saline), although this is not required. In otherinstances, a mixture of saline and a cryoprotectant (e.g., polyethyleneglycol (PEG) 3350) may be used as a diluent. The PEG concentration ofthe diluent can be approximately about 30-90 g/liter (or about 10-90g/liter). The PEG concentration of the diluent can also be approximatelybetween about 25-75 g/liter. In one example, the ratio of saline/PEGmixture to stool sample is 2:1, or 2 mL saline/PEG mixture to 1 gramhuman stool. As a non-limiting example, approximately 100 mL ofsaline/PEG mixture can be used for 50 g of human stool. In anotherexample, the ratio of saline/PEG mixture to stool sample is 3:1, or 3 mLsaline/PEG mixture to 1 gram human stool. As a non-limiting example,approximately 150 mL of saline/PEG mixture can be used for 50 g of humanstool. While saline/PEG may be suitable for use as a diluent (and/orcryoprotectant), this is not intended to be limiting. Othercryoprotectants may also be utilized. For example, dextrose, betaine,glycine, sucrose, polyvinyl alcohol, Pluronic F-127, mannitol, tween 80,ethylene glycol, 1,3-propanediol, hydroxypropyl cellulose, glycerol,PEG/glycerol mix, propylene glycol, or combinations thereof may be usedas cryoprotectants. These materials may be used alone or in combinationwith a solvent such as saline.

In one example, the sample may be placed in a 500 μm filter bag, with orwithout a diluent, and agitated using, for example, Stomacher agitationat 230 rpm for approximately 2 minutes to obtain a filtrate having aparticle size of approximately 500 μm or less. This filtrate may then beplaced in a filter bag having a pore size smaller than 500 μm, forexample, 280 μm. The sample may be agitated again using, for example,Stomacher agitation at 230 rpm with or without a diluent forapproximately 4 minutes to obtain a filtrate having a particle size ofapproximately 280 μm or less. This filtrate may be placed in anotherfilter bag having a pore size smaller than, for example, 280 μm, suchas, but not limited to 60 μm. The sample may be agitated again using,for example, Stomacher agitation at 230 rpm with or without a diluentfor approximately 4 minutes to produce a filtrate having a particle sizeof approximately 50-70 μm or less.

In another example, the sample may be placed in a 500 μm filter bag,with or without a diluent, and agitated using, for example, Stomacheragitation obtain a filtrate having a particle size of approximately 500μm or less. This filtrate may then be processed using a pressure filterhaving a pore size of approximately 160 μm and the resulting filtrateprocessed using a pressure filter having a pore size of approximately 60μm. In some instances, the sample may be need to be processed a secondtime using a bag filter having a pores size between 160 μm and 500 μmprior to using the pressure filter.

In another example, the sample may be placed in a 500 μm filter bag,with or without a diluent, and agitated using, for example, Stomacheragitation obtain a filtrate having a particle size of approximately 500μm or less. This filtrate may then be processed using a vacuum filterhaving a pore size of approximately 160 μm and the resulting filtrateprocessed using a vacuum filter having a pore size of approximately 60μm. In some instances, the sample may be need to be processed a secondtime using a bag filter having a pores size between 160 μm and 500 μmprior to using the pressure filter.

Once the sample has been processed to have a particle size ofapproximately 60 μm or less, the sample may then be washed and furtherconcentrated using a centrifuge. In some instances, centrifuge tubes mayhave a volume in the range of 50 to 500 mL, or more. The filteredsuspension is filled to approximately 20 to 80% of the volume of thecentrifuge tube. In one example, the samples may be centrifuged at 1100to 3600 revolutions per minute (rpm) for 10 to 15 minutes cycles. Inanother example, the samples may be centrifuged at a rate such that thecentrifugal force is in the range of about 8-12,000 g (e.g., about10,000 g) for 15-45 minutes or 20-30 minutes. The centrifuge may beramped up or gradually accelerated to the speed needed to create acentrifugal force in the range of about 8-12,000 g (e.g., about 10,000g). It is further contemplated that the centrifuge may also be slowlyramped down or decelerated when the centrifugation process is complete.In some instances, it may be desirable to decelerate the centrifuge asslowly as possible so that the return to atmospheric pressure is slow soas to protect the bacterial cells from potentially bursting. Thesupernatant is removed and the remaining material in the tube is thepurified intermediate MRT composition. This may result in a product thathas been concentrated by approximately 60%. In some instances, thecentrifugation process may be a 2-tiered process. For example, theproduct may first undergo a “pre-spin”, (e.g., about 300-2000×g or about1,400×g for 1-5 minutes or for about 2 minutes) to remove fecal fibrousmaterial and then may undergo a longer centrifugation to concentrate theproduct. For example, following the “pre-spin”, the supernatant may betransferred to a new centrifuge tube/bottle and then spun at a higherspeed (e.g., about 5,000-12,000×g or about 10,000×g for 30-60 minutes orfor about 45 minutes). After the high-speed spin, the supernatant may bediscarded and the recovered microbiota can be further processed. It isfurther contemplated that volumes of up to 300 mL may be centrifugedwithout resulting in a drop in the amount of concentration. Theresulting MRT composition is a bacterial suspension having a particlesize of 70 μm or less and a bacterial concentration on the order ofapproximately 1×10¹⁰ CFU/g. The resulting MRT composition may also bestable for 3 weeks at refrigeration conditions.

In some embodiments, centrifugation alone can be used multiple times forpurification and concentration. However, the particle size of thebacterial suspension may still be in a range (e.g. greater than 60 μm)that clogs pipet tips. However, in some instances, wide pipette tips maybe used. Whether this is successful or not is dependent on the inputfecal material, which is variable. It is further contemplated that asystem of separators and decanters could be used if the batch size wasin the range of several tens of liters, or more. However, this may notbe required if the starting product has been previously processed.

In other embodiments, density gradient centrifugation may be used forpurification and concentration of a fecal sample. Density gradientcentrifugation may be used in combination with the filtering techniquesdescribed above, or alone. Density gradient centrifugation may separatestrictly by density, whereas differential centrifugation may separate byparticle size and density. To perform density gradient centrifugation, adensity gradient media may be added to the sample (e.g. diluted rawsample or diluted, filtered sample). The density gradient media may be asolution of varying concentration (e.g. a sucrose having varyingconcentrations). For example, a density gradient media may be created byoverlaying lower concentrations of a solution on higher concentrationsof the solution in a centrifuge tube. The sample may be placed on top ofthe density gradient media and subsequently centrifuged. The particlesin the sample may travel through the density gradient media until theyreach the point in the gradient at which their density matches that ofthe surrounding solution. For example, the target material (e.g.bacteria) may settle in the middle of the centrifuge tube due to thedensity of the bacteria and the density gradient media. A wide varietyof density gradient media may be used for the centrifugation, including,but not limited to, polyhydric (sugar) alcohols, polysaccharides,inorganic salts, iodinated compounds, colloidal silica, etc. Otherdensity gradient materials may include iohexols such as Nycodenz®(manufactured by Axis-Shield), iodixanol solutions, such as OptiPrep™(manufactured by Axis Shield), and/or various molecular weight PEGs. Itis contemplated that concentrations in the range of 40% to 80%weight/volume (w/v) of Nycodenz® may be used. The media may bepharmaceutical grade, biologically inert, and/or isosmotic. In someinstances, density gradient centrifugation may purify bacteria fromstool more efficiently that differential centrifugation.

In some embodiments, tangential flow filtration (or crossflowfiltration) may be used in combination with density gradientcentrifugation to further remove any undesired soluble material. Intangential flow filtration, the majority of the feed flow may flowtangentially across a surface of a filter than into the filter.Tangential flow filtration of the target material (e.g. bacteria) mayfurther remove soluble impurities from the target material. During thetangential flow filtration, additional fibrous material may be pushedout as the bacterial suspension (obtained from traditionalcentrifugation and/or density gradient centrifugation) is passed acrossthe surface of the filter. In some instances, each pass of the bacterialsuspension through the tangential flow filtration system may be followedby a buffer. It is contemplated that larger volumes (e.g. up to about 10L) of bacterial suspension may be processed at one time through atangential flow filtration system. In some instances, the filtrate fromthe tangential flow filtration process may be used as the purifiedintermediate fecal sample. It is contemplated that the filteredsuspension (e.g. filtrate) may be diluted with saline and/orphosphate-buffered saline (PBS). In other instances, the filtrate fromthe tangential flow filtration process may be further processed using,for example, but not limited to, differential centrifugation and/ordead-end filtration.

In some embodiments, it may be desirable to stabilize the processedsample in suspension 104 at refrigeration conditions for a period oftime in the range of one to two weeks. In some instances, removal of thefecal material and replacement with carriers or excipients which aresoluble in an aqueous solution may allow the bacteria to be suspended inthe liquid and further processed without stability concerns.Considerations for these excipient solutions may be pH, concentration,and isotonicity or isosmolality. Excipients may be selected based onprotein and monoclonal antibody formulations and their proposed role instabilizing biologics. Some example excipients that may be used toprovide liquid stabilization 104 of the sample may include, but are notlimited to: salt (NaCl), sucrose, trehalose, L-argininemonohydrochloride, and/or PEG 3350, as summarized in Table 1 below.Lists of other potential excipients can be found in tables I and III inSeong Hoon Jeong, Arch Pharm Res Vol 35, No 11, 1871-1886, 2012 and inTables in Pramanick et al. Pharma Times, Vol 45, No. 3, March 2013.

TABLE 1 Summary of illustrative excipients. MW Excipient (g/mol)Solution % M (g/mol) NaCl 58.44 0.9 0.15 Sucrose 342.3 6 0.18 Sucrose342.3 9.25 0.27 Sucrose 342.3 12 0.35 L-Arginine 210.66 0.5 0.02Monohydrochloride L-Arginine 210.66 1.5 0.07 MonohydrochlorideL-Arginine 210.66 3 0.14 Monohydrochloride PEG 3350 3350 1 0.00 PEG 33503350 5 0.01 PEG 3350 3350 10 0.03 L-Arginine 210.66 0.17 0.01Monohydrochloride

In some instances, the excipient may include 2-20% sucrose, 0.1-5%L-arginine monohydrochloride, 0.5-20% PEG 3550, or combinations thereof.

Combinations of excipients may be used to protect biological cells ortissues from the effects of freezing and/or to provide stability (e.g.minimize cell death) to the product during storage. Table 2 belowillustrates some example excipient formulations that may providecryoprotection and stability during storage. However, the formulationslisted in Table 2 are not intended to be limiting. Other combinationsand/or quantities of excipients may also be used.

TABLE 2 Excipient Solution Compositions Prior to Adding to the DrugSubstance Component 1 Component 2 Component 3 Component 4 Component 5 #120%, PEG-120 60%, Sucrose 20%, Phosphate Methyl Glucose Buffer Solution,Dioleate pH 7.4 #2 20%, PEG-120 60%, Trehalose 20%, Phosphate MethylGlucose Buffer Solution, Dioleate pH 7.4 #3 20%, 60%, Sucrose 20%,Phosphate Polyvinylpyrrolidone Buffer Solution, (PVP) pH 7.4 #4 20%,60%, Trehalose 20%, Phosphate Polyvinylpyrrolidone Buffer Solution,(PVP) pH 7.4 #5 20%, PEG-120 20%, 40%, Sucrose 20%, Methyl GlucosePolyvinylpyrrolidone Phosphate Dioleate (PVP) Buffer Solution, pH 7.4 #620%, PEG-120 20%, 40%, Trehalose 20%, Methyl GlucosePolyvinylpyrrolidone Phosphate Dioleate (PVP) Buffer Solution, pH 7.4 #72.3% Polyethylene 10% Trehalose 10% Sucrose 1% Glycerin 76.7% PurifiedGlycol 3350 Water

In some instances, the excipient (e.g., the lyophilization excipient)may include polyethylene glycol (e.g., about 1-5%, or about 2-3%, orabout 2.3%), trehalose (e.g., about 1-25%, or about 5-15%, or about10%), sucrose (e.g., about 1-25%, or about 5-15%, or about 10%) andglycerin (e.g., about 0.1-5%, or about 0.5-2%, or about 1%) in purifiedwater.

It is contemplated that the above excipient formulations, when added tothe drug substance (e.g. fecal sample or processed fecal sample) mayprovide cryoprotection and stability during storage to the biologicalcells in a liquid and/or solid formulation. In some instances, theexcipient formulations may be added to the drug substance in a ratio of1:1. This is just an example. Other excipient to drug substance ratiosare also contemplated, for example, but not limited to 0.25:1, 0.5:1,1.5:1, 2:1, etc.

In some of these and in other instances, the excipient formulations mayinclude:

(a) 0.5-20% PEG, (b) 0.1-5% glycerin, (c) 10-30% PVP, (d) 40-80%trehalose, (e) 40-80% sucrose, (f) 10-30% phosphate buffer solution, or(g) combinations thereof. Other formulations are contemplated.

It is contemplated that similar excipients may also be used to protectthe bacteria during membrane filtration. For example, Farber and Sharpein Applied and Environmental Microbiology, Aug 1984, P. 441-443 statethat bacterial recovery is improved in the presence of certain fooddebris (carrots, cheese, peaches, tuna)—pH may be important—pH 5.88 to6.40 for carrots, pH 4.75-5.02 for cheese, pH 5.9 to 6.2 for tuna, pH3.3 to 4.05 for peaches. The presence of sugars, carbohydrates, orproteins may be important, properties of these foods that coat thebacteria, support bacterial growth (pre-biotic activity) or support thebacterial cell wall during filtration may be important.

Suitable carriers may vary with the desired form and mode ofadministration of the composition. For example, they may includediluents or excipients such as fillers, binders, wetting agents,disintegrators, surface-active agents, glidants, lubricants, and thelike. Typically, the carrier may be a solid (including powder), liquid,or combinations thereof. Each carrier is preferably “acceptable” in thesense of being compatible with the other ingredients in the compositionand not injurious to the subject. The carrier may be biologicallyacceptable and inert (e.g., it permits the composition to maintainviability of the biological material until delivered to the appropriatesite).

Oral compositions may include an inert diluent or an edible carrier. Forthe purpose of oral therapeutic administration, the active compound canbe incorporated with excipients and used in the form of tablets,troches, or capsules, e.g., gelatin capsules. Oral compositions can alsobe prepared by combining a composition of the present disclosure with afood. In one embodiment a food used for administration is chilled, forinstance, ice cream. Pharmaceutically compatible binding agents, and/oradjuvant materials can be included as part of the composition. Thetablets, pills, capsules, troches and the like can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, orange flavoring, or other suitableflavorings. These are for purposes of example only and are not intendedto be limiting.

Once the purified sample has been purified and stabilized in an aqueoussuspension which may be suitable for delivery via a gastro-nasal tube oran enema, the sample may be further processed to be suitable for an oraldelivery, such as in the form of tablets, troches, or capsules. Forexample, the aqueous solution may be converted to a solid 106. A list ofbacterial processing techniques can be found in Martin et al.,Innovative Food Science and Emerging Technologies, 27 (2015) 15-25.

In some instances, lyophilization, or freeze-drying, may be used toconvert the sample from a liquid to a solid. The sample may be providedwith a cryoprotectant such as, but not limited to PEG, skim milk,charcoal, ascorbic acid or a combination thereof to protect the bacteriafrom the effects of freezing. The sample may also be provided with alyoprotectant such as, but not limited to sucrose, inositol, trehalose,glycerol, or a combination thereof. In some instances, the sample mayalso be provided with an enrichment material which may provide acidbuffering. Alternatively or additionally, the enrichment material mayalso keep the bacteria more active which may facilitate analyticaltesting. Some example enrichment materials may include, but are notlimited to skim milk, charcoal, gelatin, ascorbic acid, GI media, orcombinations thereof. Alternatively or additionally, an oxygen scavengermay be added to the sample prior to and/or after lyophilization. Whilenot wishing to be bound by theory, it is believed that an oxygenscavenger may improve the stability and/or viability of the sample. Itis contemplated that lyophilization tubes may include an insert that canbe used to expel a lyophilized pellet from the lyophilization tube afterfreeze-drying. The width of the lyophilization tube may be smaller thanthe width of a capsule shell for oral treatment. This may allow for thedisplacement of a tray of pellets directly into the capsule shells. Itis contemplated that this may reduce or eliminate the need for particlesizing of the formulation or blending it further 108 for improvement inflow properties into the capsule. The dose may also be determined bypellet size. In some instances, a pellet produced in the lyophilizationprocess may include approximately 4.5×10⁸ CFU (CDC). A size 0 capsulemay accommodate three pellets. Thus, a capsule may include approximately6.7×10⁹ CFU (CDC). Eight capsules taken twice a day may be required tobe equivalent to one enema dose. Further, there may be no need to testfor homogeneity of the batch of pellets that are mixed together prior tocapsule filling. In some instances, tamping may allow for a greaterconcentration or number of pellets within each capsule. For example,tamping of the pellets within the capsule may allow for about 2-4 times(e.g., about 2.5 times) the number of pellets in each capsule (e.g.,without tamping each capsule may accommodate 2-4 or about 3 pelletswhereas with tamping each capsule may accommodate about 7-10 or about 8pellets). This may help to reduce the number of capsules a patient mayneed to take in order to achieve the desired dose. In some instances,the pellets may be ground prior to tamping them into the capsule. If thepellets are ground, it may be desirable for the powder to have a Carr'sIndex value in the range of 15 to 30 to facilitate capsule filling.Alternatively, the pellets may be ground and compressed into a tabletform. An enteric powder may then be pressed over the tablet to generatean oral dosage that may be stable in the acid environment of the stomachbut dissolves in the intestinal tract.

In other instances, it may be desirable to preserve the sample throughvaporization foam drying. It is contemplated that traditional excipientsand equipment may be used with this process. Higher excipientconcentrations and optimal process parameters to produce foam duringprocessing may result in low water content formulations. The lower thewater content; the greater the probability of stability at roomtemperature. Once the sample has been dried 106, the sample may befurther processed to achieve a desired particle size and/or blending 108in order to prepare the sample for oral product processing.

In yet other embodiments the liquid sample may be microencapsulated bylipids to protect from bile, alginates, and/or polymers. Once the samplehas been encapsulated, the sample may be further processed to achieve adesired particle size and/or blending 108 in order to prepare the samplefor oral product processing.

After the sample has been processed to a desired particle size and/orblended 106 in order to prepare the sample for oral product processing,the sample may be encapsulated 110. It is contemplated that theencapsulation process may provide for low pH protection 112. Forexample, the encapsulation process may prevent or substantially preventcapsule shells, tablets, and/or troches from breaking down in the acidicenvironment of the stomach such that the MRT composition is released inthe desired portion of the intestinal tract. It is contemplated that anenteric coated capsule may be needed to provide for protection in thestomach and have disintegration of the capsule in the small and largeintestine. In some instances, the capsules may be pan coated with theenteric coating. Enteric coating materials may include fatty acids,waxes, shellac, plastics, and plant fibers. Pan coating of hydroxypropylmethylcellulose (HPMC), or also called Hypromellose capsules, willprotect at low pH and also help to protect from moisture. Some suitablecapsules may include DRcaps™ and Vcaps™ available from Capsugel®.Likewise, AR caps having a composition of 60% HPMC and 40% HPMCP(hypromellose phthalate) may have the same properties. Capsule typesthat are not gelatin may contain less water (gelatin caps usually 10 to12% water, versus other polymer capsules have 3-4% or less water).Banding of the capsule with polymers that are insoluble in low pHenvironments may be required, as will be discussed in more detail below.In other instances, the capsules may be stacked such that 2 or morecapsules are used to enclose the sample. For example, the sample may beplaced in a capsule and then that capsule placed in another largercapsule. A stacked (e.g. two or more capsules) and/or banded capsule maysurvive in an acidic environment (e.g. the stomach) for at least two ormore hours and dissolve in the more neutral intestinal tract.

In some instances, a capsule that has been banded with a lowpH-resistant polymer may not fully disintegrate and/or release theproduct for 5 or more hours. This may allow the capsule to pass throughthe stomach intact and allow the product to be released into theintestines where the bacteria is desired. It is further contemplatedthat releasing the MRT composition into the more neutral environment ofthe intestines, as opposed to the acidic environment of the stomach (inthe range of a pH of 1.2) may allow more bacteria to survive. Bandingthe capsule may include placing a band of low pH-resistant polymer overthe region where the first capsule portion and the second capsuleportion overlap. In other instances, two capsules (e.g., doubleencapsulation), where one capsule is disposed within another capsule,may allow the capsule to pass through the stomach intact and/or so thata desirable quantity of viable bacteria may reach the target region.

In some embodiments, superdisintegrants may be used to expand the dosageform (e.g. capsule or tablet) to improve the probability of bacteriacontacting the intestinal wall. For example, cross-linked celluloseswells 4 to 8 times in 10 seconds, cross-linked starch swells 7 to 12times in less than 30 seconds, and cross-linked alginic acid experiencesrapid swelling in an aqueous medium or wicking action.

The presence of pre-biotics may be desired to ensure bacterial growth atsite of action in the intestine. These are materials that can be addedto the capsule formulation or dosed separately at the sameadministration time. Some suitable additives may includegalacto-oligosaccharides, inulin-derivatives such asfructo-oligosaccharides, cellulose, dextrins, chitins, pectins, beta-glucans, waxes, lignin, phytochemicals (bioactive non-nutrient plantcompounds present in fruits, vegetables, grains, and other plant foods),carotenoids, phenolics, alkaloids, nitrogen-containing and organosulfurcompounds. It is contemplated that L-arginine and PEG excipients, incertain concentration ranges, may produce water and electrolytesecretion when the drug product is delivered. This may enhance thebacteria's ability to attach and grow in the intestine. Other excipientsthat produce this effect may also improve the therapeutic effect.

An oral product may be packaged in a number of different ways including,but not limited to, blister packaging or a bottle. In some instances, anoxygen scavenger and/or a desiccant may be placed in the bottle and/orblister packaging. The blister packaging and/or bottle may includefeatures configured to make the packing child resistant. For example, abottle may be provided with a child resistant cap and the blister packmay be provided with a child resistant outer sleeve. In some instances,the blister pack may include graphics designed to guide the patient onhow to use the pack. For example, the blister pack may provide guidanceon how many pills to take on a given day and/or what time of day to takethe pills. The packaging may include monitoring devices to monitor theshipping conditions. As a non-limiting example, the packaging containerscan include an indicator of the minimum and the maximum temperature towhich the product is exposed. As another non-limiting example, one ormore temperature sensitive stickers that changes color at temperaturesbelow about 4° C. and temperatures greater than about room temperature(about 22-29° C.) can be affixed to the container.

FIGS. 3 and 4 are a flow charts depicting two illustrative methods 201,300 for preparing a stool sample for MRT as an oral dosage. In someembodiments, the oral dosage may be prepared from a fresh stool sample(FIG. 3) and in other embodiments, the oral dosage may be prepared froma substance that has already been processed (FIG. 4). As used herein afresh stool sample will be referred to as Drug Substance A and a samplethat has been previously processed will be referred to as Drug SubstanceB. Other drug substances are contemplated includes substances derivedfrom cultures of fecal microbiota. Referring first to FIG. 3, the stoolsample may first be collected and screened, for example, in the methoddescribed with respect to FIG. 1. Once the sample has been accepted, thesample may be weighed into a filtration bag, as shown at step 200. It iscontemplated that multiple collection containers (e.g. same or differentdonors and collected at various times) that are within their expirationdata may be used (e.g. pooled together). The sample may be purifiedusing centrifugation, membrane filtration, or a combination thereof toremove fecal material above a certain particle size. It is contemplatedthat since most bacteria of interest are in the range of 0.3 microns (μ)to 30 μm, the sample may be processed to remove particles greater thanin the range of 50-70 μm. The sample may be processed to obtain anapproximately 60% concentration of the bacteria. This may allow for anincreased flexibility in the ratio of formulation excipients to bacteriafor further processing.

A filter solution, or diluent, may be added to the filter bag, as shownat step 202. In some instances saline may be used as a diluent. Forexample a solution of 0.9% sodium chloride (NaCl) may be added to thefilter bag at a ratio of approximately 3 milliliters (mL) per gram ofDrug Substance A. In other instances, phosphate buffered saline may beadded to the filter bag at a ratio of 1:1 (by weight). It iscontemplated that other diluents, other diluent concentrations, anddilution rates may be used, as desired. For example, a mixture of salineand a cryoprotectant (e.g., polyethylene glycol (PEG) 3350) may be usedas a diluent. The PEG concentration of the diluent can be approximatelyabout 30-90 g/liter (or about 10-90 g/liter). The PEG concentration ofthe diluent can also be approximately between about 25-75 g/liter. Inone example, the ratio of saline/PEG mixture to stool sample is 2:1, or2 mL saline/PEG mixture to 1 gram human stool. However, in someinstances, such as when Drug Substance A is being processed specificallyfor lyophilization, the diluent may not include a cryoprotectant. Thesample may then be membrane filtered in a number of different ways,including, but not limited to the use of filter bags, pressure filters,and/or vacuum filters, as shown at step 204. In some instances, thesample may be filtered multiple times using a smaller filter membranewith each subsequent filtering. In one example, the sample may be placedin a 500 μm filter bag and agitated using, for example, Stomacheragitation at 230 rpm for approximately 2 minutes to obtain a filtratehaving a particle size of approximately 500 μm or less. This filtratemay then be placed in a filter bag having a pore size smaller than 500μm, for example, 280 μm. The sample may be agitated again using, forexample, Stomacher agitation at 230 rpm with or without a diluent forapproximately 4 minutes to obtain a filtrate having a particle size ofapproximately 280 μm or less. This filtrate may be placed in anotherfilter bag having a pore size smaller than, for example, 280 μm, suchas, but not limited to 50-70 μm. The sample may be agitated again using,for example, Stomacher agitation at 230 rpm with or without a diluentfor approximately 4 minutes to produce a filtrate having a particle sizeof approximately 50-70 μm or less.

In another example, the sample may be placed in a 500 μm filter bag,with or without a diluent, and agitated using, for example, Stomacheragitation obtain a filtrate having a particle size of approximately 500μm or less. This filtrate may then be processed using a pressure filterhaving a pore size of approximately 160 μm and the resulting filtrateprocessed using a pressure filter having a pore size of approximately 60μm. In some instances, the sample may be need to be processed a secondtime using a bag filter having a pores size between 160 μ.m and 500 μmprior to using the pressure filter.

In another example, the sample may be placed in a 500 μm filter bag,with or without a diluent, and agitated using, for example, Stomacheragitation obtain a filtrate having a particle size of approximately 500μm or less. This filtrate may then be processed using a vacuum filterhaving a pore size of approximately 160 μm and the resulting filtrateprocessed using a vacuum filter having a pore size of approximately 60μm. In some instances, the sample may be need to be processed a secondtime using a bag filter having a pores size between 160 μm and 500 μmprior to using the pressure filter.

Once the sample has been processed to have a particle size ofapproximately 50-70 μm or less, the sample may then be placed intointermediate storage containers, as shown at step 206. An example of anacceptable intermediate storage container is a 250 mL sterile plasticcontainer with lid. In some instances, the filtered suspension may bestored in the refrigerator at 5±3° C. for up to 5 days, although this isnot required. The filtered suspension may be combined and mixed intolarger containers, as shown at step 208. An example of an acceptableimmediate storage container is a multiple liter sterile plasticcontainer with lid.

Aliquots of the mixed filtered suspension may then be placed intocentrifuge tubes, 50 to 500 mL in volume, as shown at step 210. Thefiltered suspension is filled to approximately 20 to 80% of the volumeof the centrifuge tube. In some instances, centrifuge tubes having avolume of greater than 500 mL may be used. The filtered suspension maythen be washed and further concentrated using a centrifuge, as shown atstep 212. In one example, the samples may be centrifuged at 1100 to 3600revolutions per minute (rpm) for 10 to 15 minutes cycles. In anotherexample, the samples may be centrifuged at a rate such that thecentrifugal force is in the range of about 8-12,000 g (e.g., about10,000 g) for 15-45 minutes or 20-30 minutes. The centrifuge may beramped up or gradually accelerated to the speed needed to create acentrifugal force in the range of about 8-12,000 g (e.g., about 10,000g). It is further contemplated that the centrifuge may also be slowlyramped down or decelerated when the centrifugation process is complete.In some instances, it may be desirable to decelerate the centrifuge asslowly as possible so that the return to atmospheric pressure is slow soas to protect the bacterial cells from potentially bursting. Thesupernatant is removed and the remaining material in the tube is thepurified intermediate MRT composition. This may result in a product thathas been concentrated by approximately 60%.

In some instances, the centrifugation process may be a 2-tiered process.For example, the product may first undergo a “pre-spin”, (e.g., about300-2000×g or about 1,400×g for 1-5 minutes or for about 2 minutes) toremove fecal fibrous material and then may undergo a longercentrifugation to concentrate the product. For example, following the“pre-spin”, the supernatant may be transferred to a new centrifugetube/bottle and then spun at a higher speed (e.g., about 5,000-12,000×gor about 10,000×g for 30-60 minutes or for about 45 minutes). After thehigh-speed spin, the supernatant may be discarded and the recoveredmicrobiota can be further processed. It is further contemplated thatvolumes of up to 300 mL may be centrifuged without resulting in a dropin the amount of concentration. In some instances, volumes of greaterthan 300 mL may be centrifuged. For example, as discussed above, thecentrifuge volume may be selected as a percentage (for example, in therange of 60%) of the container volume. The resulting MRT composition isa bacterial suspension having a particle size of 70 μm or less and abacterial concentration on the order of approximately 1×10¹⁰ CFU/g. Thepurified intermediate bacterial viability may be measured via apropidium monoazide (PMA) quantitative polymerase chain reaction (qPCR)method. The resulting MRT composition may also be stable for 3 weeks atrefrigeration conditions.

In some embodiments, centrifugation alone can be used multiple times forpurification and concentration. However, the particle size of thebacterial suspension may still be in a range (e.g. greater than 60 μm)that clogs pipet tips. Whether this is successful or not is dependent onthe input fecal material, which is variable. It is further contemplatedthat a system of separators and decanters could be used if the batchsize was in the range of several tens of liters, or more.

The intermediate MRT composition may be optionally transferred to anintermediate tube and, if necessary, shipped to a lyophilizationfacility, as shown at step 214. Purified intermediate may be shipped ina pre-qualified shipper for refrigeration conditions, 5±3° C. to thecontract lyophilizer, if necessary, for lyophilization.

The purified intermediate may be mixed at a 1:1 ratio with alyophilization excipient solution, as shown at step 216. Thelyophilization excipient solution may be comprised of 2.3% PEG 3350, 1%glycerin, 10% trehalose, and 10% sucrose. However, other lyophilizationexcipients may be used. Prior to adding the excipient solution to thepurified intermediate, the lyophilization excipient solution (withoutglycerin) is filtered through a 0.2 μm filter. The glycerin isautoclaved at 121° C. for a minimum of 15 minutes and added aseptically.Once the lyophilization excipients and purified intermediate have beenmixed (lyophilization suspension), a single two hundred microliter (200μL) aliquot of the lyophilization suspension is placed in each well of a96-well plate, as shown at step 218 and lyophilized, as shown at step220.

The lyophilization process will be described with further reference toFIG. 5, which illustrates a flow chart of an illustrative lyophilizationprocess 220. To perform the lyophilization, once filled, the 96-wellplate may be wrapped in sterile bioshield, as shown at step 402. Otherplate sizes are also contemplated. After all plates are wrapped, theymay be immediately transported and loaded into the lyophilizer, as shownat step 404. The lyophilizer may be sealed and the lyophilization cycleinitiated. Product is frozen by lowering the product shelf temperatureto a range of approximately −40° C. to −45° C., as shown at step 406.After the product is frozen, primary drying (sublimation) occurs byapplying vacuum and elevating the shelf temperature up to 0° C., asshown at step 408. A secondary drying step is initiated to furtherreduce water content and bring the product to ambient temperature(approximately 25° C.), as shown at step 410. The vacuum is released atthe end of the secondary drying step and the product is removed from thelyophilizer, as shown at step 412. Product may be placed inside ananaerobic chamber for collection of the lyophilized aliquots. Thelyophilized aliquots may be in pellet form and are transferred to apackaging with desiccant, as shown at step 414. Filled packages may bepurged with nitrogen gas and heat-sealed, as shown at step 416.Returning now to FIG. 3, if the intermediate MRT composition has beenshipped off-site for lyophilization, the lyophilized pellets may then beshipped back to the MRT composition manufacturer, in a pre-qualifiedshipper for refrigeration conditions, as shown at step 222.

In some instances, it may be desirable for the lyophilized material orpellets to have a glass transition temperature (T_(g)) of greater than30° C. In some examples, the glass transition temperature may be in therange of 30-75° C. This may result in a final product that is stable atroom temperature. The glass transition temperature may also be used as atool for screening the product received form the lyophilization processand/or for verifying the stablility of the final product. For example,the T_(g) may be used to predict stability of the product duringstorage. In some instances, a T_(g) of 50° C. above the storagetemperature may allow the lyophilized intermediate and/or the final oraldrug product to be stored for a period of time without a significantloss of bacteria.

Upon receipt of the lyophilized intermediate, it may be removed from thepackaging and filled into capsules, as shown at step 224. Thelyophilized intermediate may also be sampled and the total viability ismeasured via a PMA-qPCR method. Encapsulation may be conducted in anitrogen-purged area at ambient temperature to minimize the exposure ofthe lyophilized intermediate to oxygen. The lyophilization intermediatesare encapsulated in a hypromellose capsule. Multiple lyophilizedintermediates can be loaded into a hypromellose capsule depending on thecapsule size (e.g., sizes 1, 0, or 00).

The capsule may then be banded, as shown at step 226. In some instances,the capsules may be banded with hypromellose. In some instances, thebanding material may be an anionic copolymer based on methacrylic acidand methyl methacrylate, such as, but not limited to Eudragit® L100. Inother instances, the banding material may be hypromellose phthalate orhypromellose acetate succinate. These are just examples. The bandingmaterial may be any material which is resistant to low pH environments(e.g. the stomach) and degrades in high pH environments (e.g. theintestinal tract). A consistent banding thickness is applied to eachcapsule so the disintegration performance meets the acceptance limit.Alternatively, the capsules may not be banded and/or are otherwise freeof a banding material. Capsules are stored at refrigeration conditions,5±3° C. in a nitrogen-purged bulk plastic container or packaged withdesiccant. Encapsulated and banded drug product may be packaged withdesiccant and heat-sealed, as shown at step 228. In some instances, theencapsulated and banded drug product may be packaged in individualdosage quantities in metallized polyester/polyethylene bonded film. Thismay minimize the exposure of the drug product to oxygen and/or moisturewhich may cause degradation of the product. The metallizedpolyester/polyethylene bonded film may have a moisture vaportransmission rate of 0.02 gr/100 in² and an oxygen transmission rate of0.0402/mL/100 in² in 24 hours. The bonded film packets may be providedto the patient in a child-resistant container to meet the need forchild-resistant clinical supply packaging. The child-resistant containermay be a 40 dram (2.5 ounces) green pharmacy vial with a child-resistantcap. The vial may be made of translucent, light resistant polypropylene.The low density polyethylene (LDPE) child-resistant cap helps preventunauthorized access by requiring that the user push down and rotate thecap to open the container.

Referring now to FIG. 4, an illustrative method 300 for preparing apreviously purified stool sample (Drug Substance B) for MRT as an oraldosage. Drug Substance B may be a fecal microbiota frozen preparation,prepared as an enema dosage form including human stool and a solution of2.3% polyethylene glycol 3350 (or other cryoprotectant) and 0.9% sodiumchloride solution for irrigation in a ratio 1 g of stool to 3 mL ofsolution. For example, Drug Substance B may have been processed in amanner similar to steps 200 through 212 described above, with theaddition of a cryoprotectant at step 202. After the centrifugationprocess outlined at step 212, the purified intermediate (e.g. now DrugSubstance B) may be refrigerated, frozen, or used for treatment.

Beginning at step 302,the frozen preparation may be thawed, ifnecessary, and placed into a filtration bag. It is contemplated thatmultiple collection containers (e.g. same or different donors andcollected at various times) that are within their expiration data may beused. The sample may be purified using centrifugation, membranefiltration, or a combination thereof to remove fecal material above acertain particle size. It is contemplated that since most bacteria ofinterest are in the range of 0.3 microns (μm) to 30 μm, the sample maybe processed to remove particles greater than in the range of 50-70 μm.The sample may be processed to obtain an approximately 60% concentrationof the bacteria. This may allow for an increased flexibility in theratio of formulation excipients to bacteria for further processing.

A filter solution, or diluent, may be added to the filter bag, as shownat step 304. In some instances saline may be used as a diluent. Forexample a solution of 0.9% sodium chloride (NaCl) may be added to thefilter bag at a ratio of approximately 3 milliliters (mL) per gram ofDrug Substance B. It is contemplated that other diluents, other diluentconcentrations, and dilution rates may be used, as desired. The samplemay then be membrane filtered in a number of different ways, including,but not limited to the use of filter bags, pressure filters, and/orvacuum filters, as shown at step 306. In some instances, the sample maybe filtered multiple times using a smaller filter membrane with eachsubsequent filtering. In one example, the sample may be placed in a 500μm filter bag and agitated using, for example, Stomacher agitation at230 rpm for approximately 2 minutes to obtain a filtrate having aparticle size of approximately 500 μm or less. This filtrate may then beplaced in a filter bag having a pore size smaller than 500 μm, forexample, 280 μm. The sample may be agitated again using, for example,Stomacher agitation at 230 rpm with or without a diluent forapproximately 4 minutes to obtain a filtrate having a particle size ofapproximately 280 μm or less. This filtrate may be placed in anotherfilter bag having a pore size smaller than, for example, 280 μm, suchas, but not limited to 50-70 μm. The sample may be agitated again using,for example, Stomacher agitation at 230 rpm with or without a diluentfor approximately 4 minutes to produce a filtrate having a particle sizeof approximately 50-70 μm or less.

In another example, the sample may be placed in a 500 μm filter bag,with or without a diluent, and agitated using, for example, Stomacheragitation obtain a filtrate having a particle size of approximately 500μm or less. This filtrate may then be processed using a pressure filterhaving a pore size of approximately 160 μm and the resulting filtrateprocessed using a pressure filter having a pore size of approximately 60μm. In some instances, the sample may be need to be processed a secondtime using a bag filter having a pores size between 160 μm and 500 μmprior to using the pressure filter.

In another example, the sample may be placed in a 500 μm filter bag,with or without a diluent, and agitated using, for example, Stomacheragitation obtain a filtrate having a particle size of approximately 500μm or less. This filtrate may then be processed using a vacuum filterhaving a pore size of approximately 160 μm and the resulting filtrateprocessed using a vacuum filter having a pore size of approximately 60μm. In some instances, the sample may be need to be processed a secondtime using a bag filter having a pores size between 160 μm and 500 μmprior to using the pressure filter.

Once the sample has been processed to have a particle size ofapproximately 50-70 μm or less, the sample may then be placed intointermediate storage containers, as shown at step 308. An example of anacceptable intermediate storage container is a 250 mL sterile plasticcontainer with lid. In some instances, the filtered suspension may bestored in the refrigerator at 5±3° C. for up to 5 days, although this isnot required. The filtered suspension may be combined and mixed intolarger containers, as shown at step 310. An example of an acceptableimmediate storage container is a multiple liter sterile plasticcontainer with lid.

Aliquots of the mixed filtered suspension may then be placed intocentrifuge tubes, 50 to 500 mL in volume, as shown at step 312. Thefiltered suspension is filled to approximately 20 to 80% of the volumeof the centrifuge tube. In some instances, centrifuge tubes having avolume of greater than 500 mL may be used. The filtered suspension maythen be washed and further concentrated using a centrifuge, as shown atstep 314. In one example, the samples may be centrifuged at 1100 to 3600revolutions per minute (rpm) for 10 to 15 minutes cycles. In anotherexample, the samples may be centrifuged at a rate such that thecentrifugal force is in the range of about 8-12,000 g (e.g., about10,000 g) for 15-45 minutes or 20-30 minutes. The centrifuge may beramped up or gradually accelerated to the speed needed to create acentrifugal force in the range of about 8-12,000 g (e.g., about 10,000g). It is further contemplated that the centrifuge may also be slowlyramped down or decelerated when the centrifugation process is complete.In some instances, it may be desirable to decelerate the centrifuge asslowly as possible so that the return to atmospheric pressure is slow soas to protect the bacterial cells from potentially bursting. Thesupernatant is removed and the remaining material in the tube is thepurified intermediate MRT composition. This may result in a product thathas been concentrated by approximately 60%.

In some instances, the centrifugation process may be a 2-tiered process.For example, the product may first undergo a “pre-spin”, (e.g., about300-2000×g or about 1,400×g for 1-5 minutes or for about 2 minutes) toremove fecal fibrous material and then may undergo a longercentrifugation to concentrate the product. For example, following the“pre-spin”, the supernatant may be transferred to a new centrifugetube/bottle and then spun at a higher speed (e.g., about 5,000-12,000×gor about 10,000×g for 30-60 minutes or for about 45 minutes). After thehigh-speed spin, the supernatant may be discarded and the recoveredmicrobiota can be further processed. It is further contemplated thatvolumes of up to 300 mL may be centrifuged without resulting in a dropin the amount of concentration. In some instances, volumes of greaterthan 300 mL may be centrifuged. For example, as discussed above, thecentrifuge volume may be selected as a percentage (for example, in therange of 60%) of the container volume. The resulting MRT composition isa bacterial suspension having a particle size of 70 μm or less and abacterial concentration on the order of approximately 1×10¹⁰ CFU/g. Thepurified intermediate bacterial viability may be measured via apropidium monoazide (PMA) quantitative polymerase chain reaction (qPCR)method. The resulting MRT composition may also be stable for 3 weeks atrefrigeration conditions.

In some embodiments, centrifugation alone can be used multiple times forpurification and concentration. However, the particle size of thebacterial suspension may still be in a range (e.g. greater than 60 μm)that clogs pipet tips. Whether this is successful or not is dependent onthe input fecal material, which is variable. It is further contemplatedthat a system of separators and decanters could be used if the batchsize was in the range of several tens of liters, or more.

The intermediate MRT composition may be optionally transferred to anintermediate tube and, if necessary, shipped to a lyophilizationfacility, as shown at step 316. Purified intermediate may be shipped ina pre-qualified shipper for refrigeration conditions, 5±3° C. to thecontract lyophilizer, if necessary, for lyophilization.

The purified intermediate may be mixed at a 1:1 ratio with alyophilization excipient solution, as shown at step 318. Thelyophilization excipient solution may be comprised of 2.3% PEG 3350, 1%glycerin, 10% trehalose, and 10% sucrose. However, other lyophilizationexcipients may be used. Prior to adding the excipient solution to thepurified intermediate, the lyophilization excipient solution (withoutglycerin) is filtered through a 0.2 μm filter. The glycerin isautoclaved at 121° C. for a minimum of 15 minutes and added aseptically.Once the lyophilization excipients and purified intermediate have beenmixed (lyophilization suspension), a single two hundred microliter (200μL) aliquot of the lyophilization suspension is placed in each well of a96-well plate, as shown at step 320 and lyophilized, as shown at step322.

The purified intermediate may be mixed at a suitable ration (e.g., a 1:1ratio) with a lyophilization excipient solution, as shown at step 318.In some instances, the lyophilization excipient may include polyethyleneglycol (e.g., about 1-5%, or about 2-3%, or about 2.3%), trehalose(e.g., about 1-25%, or about 5-15%, or about 10%), sucrose (e.g., about1-25%, or about 5-15%, or about 10%) and glycerin (e.g., about 0.1-5%,or about 0.5-2%, or about 1%) in purified water. For example, thelyophilization excipient solution may be comprised of 2.3% PEG 3350, 1%glycerin, 10% trehalose, and 10% sucrose. However, other lyophilizationexcipients may be used. Prior to adding the excipient solution to thepurified intermediate, the lyophilization excipient solution (withoutglycerin) is filtered through a 0.2 μm filter. The glycerin isautoclaved at 121° C. for a minimum of 15 minutes and added aseptically.Once the lyophilization excipients and purified intermediate have beenmixed (lyophilization suspension), a single two hundred microliter (200μL) aliquot of the lyophilization suspension is placed in each well of a96-well plate, as shown at step 320 and lyophilized, as shown at step322.

Another example process may include manufacturing an MRT compositionsuitable for use as a drug administered via enema and/or suitable foruse as the starting material for manufacturing a drug for oraladministration. The process may include collecting a fresh human fecalsample from a pre-screened donor. Such processes may be similar to thosedisclosed herein and/or similar to those disclosed in U.S. Pat. Nos.9,675,648 and 9,629,881, which are herein incorporated by reference. Insome instances, multiple samples from the same donor are collected andpooled. The pooled sample, which may also be termed the drug substance,may be stored at 5° C.±3° C. in a sterile microbiology container. One ormore additional samples, and/or portions of the pooled sample, may bestored as a reserve samples in a sterile microbiology container in a−80° C. freezer.

A portion of the pooled sample/drug substance (e.g., 50±10 g) may betaken from the pooled sample and disposed in a filter bag assembly. Thefilter bag assembly may include a filter bag within an outer closurebag. An excipient solution (e.g., which also may be understood to be adiluent, cryoprotectant, or other solution) may be added to the drugsubstance. The excipient solution may include 10-90 g/L, or about 20-50g/L or about 30g/L polyethylene glycol (e.g., polyethylene glycol 3350powder) in 0.9% sodium chloride. The excipient solution may be added ata suitable ratio such as 1-5 mL per 1 g of drug substance (e.g., 3 mL ofthe excipient solution per 1 g of drug substance). The process ofplacing a portion/sample of pooled sample/drug substance into a filterbag assembly, followed by addition of the excipient solution, may berepeated until the pooled sample/drug substance is sufficiently used up.For example, if the weight of the remainder of the pooled sample/drugsubstance is 50±10 g or more, another sample of pooled sample/drugsubstance may be added to another filter bag assembly. Once the weightof the remainder of the pooled sample is less than 50±10 g, theremainder of the pooled sample/drug substance may be discarded and thefilter bag assemblies may be closed.

One at a time, closed filter bag assemblies containing drug substanceand excipient solution may be mixed. For example, the filter bagassemblies may be placed into the paddle mixer and processed (e.g., forabout 2 minutes at a speed of about 230 RPM). The paddle mixer run timeand speed may be electronically controlled, and the settings may beverified prior to manufacturing every batch.

The first filter bag assembly processed in the paddle mixer may beopened and the filtrate may be withdrawn and filled into cryovials. Thecryovials can be submitted to quality control (QC) and stored in a −80°C. freezer. Quality Control drug product release samples may be testedin a QC laboratory. Reserve samples may be stored in a −80° C. freezer.

The fill tube cap of an ethylene vinyl acetate (EVA) enema bag may beremoved and 150±30 g of the microbiota suspension (e.g., the filtereddrug substance and excipient) may be withdrawn from the filter bagassembly and filled into the EVA bag through the fill port. When fillingis complete, the fill tube cap is replaced, sealing the EVA bag prior toremoval from the biosafety cabinet. The fill tube on the EVA bag may besealed between the bag and fill cap using a tube sealer to preventinadvertent opening of the container-closure. A tag labeled with drugproduct batch number, and a “quarantine” batch status sticker may beattached to every EVA enema bag.

The in-process drug product may be refrigerated at 5° C.±3° C. prior tofreezing in a -80° C. freezer. The drug product can be held at 5° C.±3°C. for up to 24 hours prior to freezing in a −80° C. freezer. The drugproduct (e.g., contained within the sealed enema bag) may be transferredfrom refrigerated storage to a designated −80° C. drug productquarantine freezer. Quarantined drug product remains in this locationuntil it is dispositioned by QC. If all donor and QC test results areacceptable the batch will be dispositioned as released. If results arenot acceptable the batch will be dispositioned as rejected anddiscarded. Drug product dispositioned as accepted will be removed fromthe −80° C. quarantine freezer, labelled “Accepted” and transferred to adesignated released drug product −80° C. freezer. The accepted, releaseddrug product, which may be similar to Drug Substance B, may be thawedand administered to a patient (e.g., via enema). The released drugproduct may also be understood to be the suspended intermediate,suitable for use in manufacturing an oral MRT composition as describedbelow.

Batch manufacturing of an oral MRT composition begins with selection ofmultiple bags of released drug product (e.g., where each bag of releaseddrug product contains a frozen suspended intermediate manufactured asdescribed above). Each of the bags of released drug product may be fromthe same donor. The selected bags may be thawed at a suitabletemperature (e.g., room temperature) for a suitable time (e.g., about 2hours) and a quantity (e.g., 500 g) of the suspended intermediate may betransferred into one or more 1-liter centrifuge bottles. A diluent maybe added to the suspending intermediate. The diluent may be phosphateBuffered Saline (PBS) or another suitable diluent. The diluent may beadded to the suspended intermediate in a suitable ratio (e.g., 1:1 ratioby weight), mixed by intermittent gentle shaking, and held for 30minutes at 4° C. At this point, the sample may be termed the dilutedintermediate.

The diluted intermediate may be differentially centrifuged. For example,the diluted intermediate may be centrifuged at a relatively low speed.For example, the diluted intermediate may be centrifuged for about 1-5minutes (e.g., 2 min) at about 500-2,000 ×g, or about 1,000-1,500×g, orabout 1,400×g. The low speed centrifugation may occur at 4° C.±3° C. Thesupernatant from this slow speed spin may be transferred into one ormore new 1 L centrifuge bottles (e.g., containing bottle liners) forfurther processing, and the pelleted material may be discarded. Thebottles containing the collected supernatant may then centrifuged at arelatively high speed. For example, the collected supernatant may becentrifuged at about 5,000-20,000, or about 8,000-12,000×g, or about10,000×g for about 15-60 minutes (e.g., about 45 minutes). The highspeed centrifugation may occur at 4° C.±3° C. After the high-speed spin,the supernatant may discarded and the remaining pellet (e.g., which maybe pellets if multiple bottles are centrifuged) are retained for furtherprocessing. The pellet(s) may be termed the recovered microbiota.

A lyophilization excipient/cryoprotectant may be added to the recoveredmicrobiota in at a suitable ratio. For example, the lyophilizationexcipient may be added to the recovered mircrobiota at a 1:1 ratio(w/w). In some instances, the lyophilization excipient may includepolyethylene glycol (e.g., about 1-5%, or about 2-3%, or about 2.3%),trehalose (e.g., about 1-25%, or about 5-15%, or about 10%), sucrose(e.g., about 1-25%, or about 5-15%, or about 10%) and glycerin (e.g.,about 0.1-5%, or about 0.5-2%, or about 1%) in purified water. Forexample, the lyophilization excipient solution may be comprised of 2.3%PEG 3350, 1% glycerin, 10% trehalose, and 10% sucrose. The mixturecontained in the bottle liner may be mixed using a paddle mixer to forma uniform suspension. The resuspended microbiota solution may bealiquoted into 96-well plates (e.g., 200 μl per well) and subjected to alyophilization process. Other plate type may be utilized and/or a singlewell dish.

An example lyophilization process will be described with furtherreference to FIG. 5, which illustrates a flow chart of an illustrativelyophilization process 220/322. To perform the lyophilization, oncefilled, the 96-well plate may be wrapped in sterile bioshield, as shownat step 402. Other plate sizes are also contemplated. In someembodiments, a tray having zero wells may also be used. This maymaximize the volume available to receive the lyophilized suspension,which may increase efficiency in the lyophilization process. After allplates are wrapped, they may be immediately transported and loaded intothe lyophilizer, as shown at step 404. The lyophilizer may be sealed andthe lyophilization cycle initiated. Product is frozen by lowering theproduct shelf temperature to a range of approximately −40° C. to −45°C., as shown at step 406. After the product is frozen, primary drying(sublimation) occurs by applying vacuum and elevating the shelftemperature up to 0° C., as shown at step 408. A secondary drying stepis initiated to further reduce water content and bring the product toambient temperature (approximately 25° C.), as shown at step 410. Thevacuum is released at the end of the secondary drying step and theproduct is removed from the lyophilizer, as shown at step 412. Productmay be placed inside an anaerobic chamber for collection of thelyophilized aliquots. The lyophilized aliquots may be in pellet form andare transferred to a packaging with desiccant, as shown at step 414.Filled packages may be purged with nitrogen gas and heat-sealed, asshown at step 416. If the intermediate MRT composition has been shippedoff-site for lyophilization, the lyophilized pellets may then be shippedback to the MRT composition manufacturer, in a pre-qualified shipper forrefrigeration conditions, as shown at step 324. In some instances, thelyophilized pellets may be termed the lyophilized intermediate.

In some instances, the composition of the lyophilized intermediate (%w/w) may include the processed microbiota (e.g., about 10-75%, or about40-60%, or about 45-50%, or about 47.9%), polyethylene glycol (e.g.,about 1-10%, or about 3-8%, or about 5.2%), glycerin (e.g., about0.5-5%, or about 1-4%, or about 2.2%), trehalose (e.g., about 10-40%, orabout 20-30%, or about 22.4%), and sucrose (e.g., about 10-40%, or about20-30%, or about 22.4%). The processed microbiota may include about1×10⁵ to 1×10¹² viable bacteria, or about 1×10⁶ to 133 10¹¹ viablebacteria, or about 1×10⁷ to 1×10¹⁰ viable bacteria. It can beappreciated that the composition of the lyophilized intermediate, whichmay be mechanically processed prior to being placed into a capsule,represents the composition of the active ingredient of the oral MRTcomposition. The disclosed compositions result when the manufacturingprocess utilizes a lyophilization excipient that includes polyethyleneglycol (e.g., about 1-5%, or about 2-3%, or about 2.3%), trehalose(e.g., about 1-25%, or about 5-15%, or about 10%), sucrose (e.g., about1-25%, or about 5-15%, or about 10%) and glycerin (e.g., about 0.1-5%,or about 0.5-2%, or about 1%) in purified water.

In some instances, it may be desirable for the lyophilized pellets tohave a glass transition temperature (T_(g)) of greater than 30° C. Insome examples, the glass transition temperature may be in the range of30-75° C. This may result in a final product that is stable at roomtemperature. The glass transition temperature may also be used as a toolfor screening the product received form the lyophilization processand/or for verifying the stablility of the final product. For example,the T_(g) may be used to predict stability of the product duringstorage. In some instances, a T_(g) of 50° C. above the storagetemperature may allow the lyophilized intermediate and/or the final oraldrug product to be stored for a period of time without a significantloss of bacteria.

Upon receipt of the lyophilized intermediate, it may be removed from thepackaging, milled or otherwise broken into smaller particles and/or apowder-like consistency, and the milled material may be filled intocapsules, as shown at step 326. The lyophilized intermediate may also besampled and the total viability is measured via a PMA-qPCR method (e.g.,example methods are disclosed in U.S. Patent Application Pub. No. US2017/0327862, which is herein incorporated by reference). Encapsulationmay be conducted in a nitrogen-purged area at ambient temperature tominimize the exposure of the lyophilized intermediate to oxygen. Thelyophilization intermediates are encapsulated in one or morehypromellose capsules. Multiple lyophilized intermediates (e.g. multiplepellets) can be loaded into a hypromellose capsule depending on thecapsule size (e.g., sizes 1, 0, or 00). For example, the milled drugproduct may be encapsulated in a size 0 capsule using manual capsulefilling equipment within an NF-grade nitrogen purged glove box. Contentuniformity among the capsules may be tested for the filled capsules.These capsules may be then filled into size 00 capsules. In other words,the drug product may be double encapsulated.

Optionally, the capsule may then be banded, as shown at step 328. Insome instances, the capsules may be banded with hypromellose. In someinstances, the banding material may be Eudragit L100, hypromellosephthalate, or hypromellose acetate/succinate. These are just examples.The banding material may be any material which is resistant to low pHenvironments (e.g. the stomach) and degrades in high pH environments(e.g. the intestinal tract). A consistent banding thickness is appliedto each capsule so the disintegration performance meets the acceptancelimit. Capsules are stored at refrigeration conditions, 5±3° C. in anitrogen-purged bulk plastic container or packaged with desiccant. Thecapsules may be packaged with desiccant and heat-sealed, as shown atstep 330. In some instances, the capsules may be packaged in individualdosage quantities in metallized polyester/polyethylene bonded film. Thismay minimize the exposure of the drug product to oxygen and/or moisturewhich may cause degradation of the product. The metallizedpolyester/polyethylene bonded film may have a moisture vaportransmission rate of 0.02 gr/100 in² and an oxygen transmission rate of0.0402/mL/100 in² in 24 hours. The bonded film packets may be providedto the patient in a child-resistant container to meet the need forchild-resistant clinical supply packaging. The child-resistant containermay be a 40 dram (2.5 ounces) green pharmacy vial with a child-resistantcap. The vial may be made of translucent, light resistant polypropylene.The low density polyethylene (LDPE) child-resistant cap helps preventunauthorized access by requiring that the user push down and rotate thecap to open the container.

In at least some instances, the moisture content of the encapsulateddrug product (e.g., including the double encapsulated drug product) isless than or equal to about 10%, or about less than or equal to about8%, or less than or equal to about 6%. In at least some instances, theencapsulated drug product is packaged to limit/minimize any furthermoisture update.

In at least some instances, the drug product may include about 10% ormore bacteria from the class Bacteroidia, or about 15% or more bacteriafrom the class Bacteroidia, or about 20% or more bacteria from the classBacteroidia.

EXAMPLES

The disclosure may be further clarified by reference to the followingExamples, which serve to exemplify some embodiments, and not to limitthe disclosure.

Example 1 Determination of Collapse Temperatures for MRT SampleFormulations

The collapse temperature results for twelve sample microbiotiarestorative therapy formulations were identified. The collapsetemperature may be used to assist in developing optimal formulations andlyophilization cycle parameters to freeze-dry this type of product in areasonable amount of time without compromising its physical or chemicalintegrity. A standard lyophilization cycle was executed for theseformulations and contained anaerobic microbial cell suspensions.

Example 2 Materials and Methods for Freeze Dry Microscopy

Twelve formulations were utilized for testing. Each base consisted ofskim milk 10%, ascorbic acid 1%, gelatin 1.4% and charcoal 0.3%.Ingredients were food grade, USP or NF grade chemicals. The base wasthen supplemented with each of the following additives:

Trehalose 10% and Sucrose 10%

Sucrose 10% and Inositol 5%

Trehalose 10% and Glycerol 1%

Raffinose 10% and Inositol 5%

Raffinose 10% and Glycerol 1%

Glucose 5% and Inositol 5%

PEG 1% and Sucrose 10%

PEG 1% and Glycerol 1%

Trehalose 10%, Sucrose 10% and Glycerol 1%

Sucrose 10% and Lactose 8%

Trehalose 10% and Inositol 5%

PEG 1% and Lactose 8%

-   The formulations were prepared. The freeze-dry microscopy instrument    consisted of a Olympus BX53 polarized light microscope with a Linkam    FDCS196 thermal stage, a T 95 system controller, a LNP liquid    nitrogen pump, and an Edwards E2M1.5 vacuum pump.

A 20 microliter (μL) aliquot of a 100 milliliter (ml) sample was placedon a glass slide which had been placed on the thermal stage afterapplying a small drop of silicone oil. A small coverslip was placed overthe sample and the chamber was sealed. The sample was then cooled to −45degree Celsius (° C.) at 10° C./minute. The temperature at which thematerial became frozen during the cooling stage was recorded. Once thetemperature dropped to −45° C., the vacuum was initiated. The productsample was then warmed at 1° C./minute. The product sample was monitoredcontinuously during the cycle to observe the drying and sublimationfronts. Once evidence of collapse was observed the temperature wasrecorded. Table 3 is a summary of the freezing temperature and thecollapse temperature for each of the formulations.

TABLE 3 Freezing temperatures and collapse temperatures recorded foreach formulation. Freezing Collapse Formulation Temperature TemperatureTrehalose and Sucrose −20° C. −24° C. Sucrose and Inositol −15° C. −20°C. Trehalose and Glycerol −16° C. −24° C. Raffinose and Inositol −22° C.−22° C. Raffinose and Glycerol −18° C. −26° C. Glucose and Inositol −13°C. −23° C. PEG and Sucrose −11° C. −23° C. PEG and Glycerol −12° C. −22°C. Trehalose, Sucrose and Glycerol −16° C. −26° C. Sucrose and Lactose−17° C. −25° C. Trehalose and Inositol −16° C. −25° C. PEG and Lactose−17° C. −20° C.

Lyophilization cycles are influenced by a variety of factors includingpercent solids in the formulations, vial size and diameter, collapsetemperatures, chamber pressures, shelf temperatures, product resistance,etc. The chamber pressure and shelf temperature necessary to completethe primary drying process is determined by the thermal characteristicsof the formulation, mainly the collapse temperature. The primary dryingtemperature is colder than the collapse temperature to account forproduct warming that occurs from increased resistance from the growingdried layer. Three cycles were designed based on the combination offactors above. All cycle times were less than 48 hours to complete.Table 4 is a summary of the drying temperature and chamber pressures forthe lyophilization cycles based on critical collapse temperatures.

TABLE 4 Primary drying temperatures and chamber pressures for theLyophilization cycles based on critical collapse temperatures. CriticalPrimary Drying Chamber Temperature Temperature Pressure −20° C. to −22°C. −30° C. 120 mTorr −23° C. to −24° C. −33° C.  95 mTorr −25° C. to−26° C. −35° C.  75 mTorr

A lyophilization cycle was designed based on data collected during thefreeze dry microscopy studies. A pilot lyophilization cycle wasconducted for each of the formulations to test for cake structure andsurvival of a bacterial cell mixture. Harvesting of cells and dispensingof the suspension were completed based on protocols established byGibson Bioscience to obtain microbial ranges of 10e7 to 10e8 colonyforming units per 100 microliter aliquot of the mix. The microorganismstocks used were selected from the first phase of the study and includedthe following anaerobes: Bacteroides uniformis ATCC 8492, Alistipesputredinis ATCC 29800, Ruminococcus gnavus ATCC 29149 and Bacteroidesovatus ATCC 8484.

The number of viable cells (CFU) before and after lyophilization wasdetermined by serial dilution method. Dilutions consisted of thefollowing levels: 10e3, 10e5, 10e7, and 10e9. Pellet samples wererehydrated in 1 mL of Phosphate Buffered Saline. All samples were platedto pre-reduced CDC Anaerobic Blood Agar and selective Bacteroides BileEsculin Agar in duplicate. Agar plates were incubated at 35-37° C. for48 hours in an anaerobic atmosphere.

The lyophilization cycles produced good quality cake structures for allformulations. Pellets were solid and uniform in appearance. Eachlyophilized pellet dissolved within 30 seconds upon rehydration in 1.0mL of Phosphate Buffered Saline. Survival rates were calculated as apercentage of the total number of bacterial colony forming units afterfreeze-drying divided by the total number of bacterial colony formingunits before freeze-drying. Colony Forming Units were based on the mixof the 4 organisms. The viability and percent survival of total colonyforming units for each formulation are summarized in Tables 5 and 6.

TABLE 5 Viability and percent survival of total colony forming units foreach formulation inoculated directly to CDC Anaerobic Blood Agar. TotalCFU Pre- Total CFU Post- Percent Formulation LyophilizationLyophilization Survival* Trehalose, Sucrose, 2.10E+08 9.05E+07 95.61%Glycerol Trehalose, Inositol 5.65E+08 5.00E+07 87.97% Sucrose, Lactose1.70E+08 1.30E+08 98.58% Trehalose, Sucrose 5.15E+08 4.95E+08 99.80%Sucrose, Inositol 6.30E+07 6.00E+07 99.73% Raffinose, Inositol 3.40E+082.85E+08 99.10% Trehalose, Glycerol 3.00E+08 1.30E+08 95.72% Raffinose,Glycerol 3.65E+08 2.25E+08 97.55% PEG, Sucrose 1.05E+08 6.10E+07 97.23%Glucose, Inositol 2.17E+09 4.05E+08 92.19% PEG, Lactose 3.10E+082.40E+08 98.69% PEG, Glycerol 4.15E+08 3.25E+08 98.77% *Based on LogTransformed Data

TABLE 6 Viability and percent survival of total colony forming units foreach formulation inoculated directly to selective Bacteroides BileEsculin Agar. Total CFU Total CFU Percent Formulation Pre-Lyo Post-LyoSurvival* Trehalose, Sucrose, Glycerol 1.26E+07 2.75E+06 90.69%Trehalose, Inositol 1.95E+08 4.05E+05 67.64% Sucrose, Lactose 1.37E+071.00E+05 70.06% Trehalose, Sucrose 2.45E+07 2.42E+07 99.92% Sucrose,Inositol 1.35E+07 1.02E+07 98.29% Raffinose, Inositol 7.50E+07 1.00E+0676.19% Trehalose, Glycerol 8.05E+07 7.50E+05 74.31% Raffinose, Glycerol3.00E+07 1.00E+04 53.50% PEG, Sucrose 1.05E+07 5.55E+05 81.81% Glucose,Inositol 4.50E+07 9.00E+05 77.80% PEG, Lactose 1.25E+07 1.50E+05 72.93%PEG, Glycerol 7.10E+07 1.15E+04 51.72% *Based on Log Transformed Data

Based on the data collected, the selected anaerobes showed the highestsurvival rate when the combination of Trehalose and Sucrose or Sucroseand Inositol were utilized in the base formulation. This was true forrecovery on both CDC Anaerobic Blood Agar and selective Bacteroides BileEsculin Agar. These results indicate that the combination of Trehaloseand Sucrose or Sucrose and Inositol provide the best protection forBacteroides sp. during lyophilization.

Example 3 Bacterial Stability of Solid Product During Storage

A study was preformed to determine the stability of the packagedencapsulated capsule after manufacturing and upon storage. A standardmicrobiological plating method, a molecular non-culture PMA-qPCR method,and 16 s rRNA gene sequencing of both PMA and non-PMA treated sampleswere employed to characterize the active component (bacteria) present inthe solid drug product. The plating and total viability stability dataindicate that a lyophilized packaged, encapsulated product (using afirst lyophilization process) is more stable at colder storageconditions (5±3° C.) than at higher storage temperatures and relativehumidity (25±2° C./60%±5% RH and 30±2° C./65%±5% RH). The plating andtotal viability stability data for a lyophilized packaged, encapsulatedproduct (using a second lyophilization process) indicates that packaged,encapsulated product is stable at both 5±3° C. and 25±2° C. storagetemperatures.

Example 4 Example Process for Manufacturing an MRT Composition Suitablefor Use as a Drug Administered Via Enema and/or Suitable for Use as theStarting Material for Manufacturing a Drug for Oral Administration

NOTE: Steps 1-3 may be performed within a Type II Class B2 biosafetycabinet. All instruments used to manipulate drug substance, excipientsolution or drug product formulation (i.e. tongue depressors,serological pipettes) are single use instruments provided pre-sterilizedby the manufacturer. Filter bags, closure bags and EVA bags are alsoprovided pre-sterilized by the manufacturer.

Step 1—Collect Donor Pooled Drug Substance Test Sample and DrugSubstance Reserve Samples

A fresh human fecal sample can be collected from a pre-screened donor.In some instances, multiple samples from the same donor are collectedand pooled. The pooled sample may be stored at 5° C.±3° C. in a sterilemicrobiology container. One or more additional samples, and/or portionsof the pooled sample, may be stored as a reserve samples in a sterilemicrobiology container in a −80° C. freezer.

Step 2—Dispense Drug Substance

A 50±10 g sample (e.g., the drug substance) may be taken from the pooledsample and disposed in a filter bag assembly. The filter bag assemblymay include a filter bag within an outer closure bag.

Step 3—Dispense Excipient Solution

An excipient solution (e.g., which also may be understood to be adiluent, cryoprotectant, or other solution) may be added to the drugsubstance. The excipient solution may include 30 g/L polyethylene glycol(e.g., polyethylene glycol 3350 powder) in 0.9% sodium chloride. Theexcipient solution may be added at a ratio of 3 mL of the excipientsolution per 1 g of drug substance. If the weight of the remainder ofthe pooled sample is 50±10 g or more, steps 2 and 3 may be repeated. Ifthe weight of the remainder of the pooled sample is less than 50±10 g,the remainder of the pooled sample may be discarded and the filter bagassemblies may be closed.

Step 4—Mixing and Filtration

One at a time, closed filter bag assemblies containing drug substanceand excipient solution are placed into the paddle mixer and processedfor 2 minutes at a speed of 230 RPM. The paddle mixer run time and speedare electronically controlled, settings are verified prior tomanufacturing every batch.

NOTE: Steps 5-6 are performed within a Type II Class B2 biosafetycabinet.

Step 5—Collect Drug Product Quality Control and Reserve Samples

The first filter bag processed in the paddle mixer is opened and thefiltrate may be withdrawn and filled into cryovials. The cryovials canbe submitted to quality control (QC) and stored in a −80° C. freezer. QCsamples are only collected from the first dose manufactured for eachbatch.

Step 5A—Drug Product Quality Control Release Testing

Quality Control drug product release samples are tested in a QClaboratory. Reserve samples may be stored in a −80° C. freezer.

Step 6—Filling

The fill tube cap of an ethylene vinyl acetate (EVA) enema bag may beremoved and 150±30 g of the microbiota suspension (e.g., the filtereddrug substance and excipient) is withdrawn from the filter bag assemblyand filled into the EVA bag through the fill port. When filling iscomplete, the fill tube cap is replaced, sealing the EVA bag prior toremoval from the biosafety cabinet.

Step 7—Seal EVA Bag

The fill tube on the EVA bag may be sealed between the bag and fill capusing a tube sealer to prevent inadvertent opening of thecontainer-closure.

Step 8—Attach Batch Identification Tags and Quarantine Labels

A tag labeled with drug product batch number, and a “quarantine” batchstatus sticker is attached to every EVA enema bag.

Step 9—Refrigerate Drug Product at 5° C.±3° C.

The in-process drug product is refrigerated at 5° C.±3° C. prior tofreezing in a −80° C. freezer. The drug product can be held at 5° C.±3°C. for up to 24 hours prior to freezing in a −80° C. freezer.

Step 10—Freeze Drug Product in a −80° C. Freezer

The drug product (e.g., contained within the sealed enema bag) may betransferred from refrigerated storage to a designated −80° C. drugproduct quarantine freezer. Quarantined drug product remains in thislocation until it is dispositioned by QC.

Step 11—Drug Product Disposition

If all donor and QC test results are acceptable the batch will bedispositioned as released. If results are not acceptable the batch willbe dispositioned as rejected and discarded.

Step 12—Release Drug Product

Drug product dispositioned as accepted will be removed from the −80° C.quarantine freezer, labelled “Accepted” and transferred to a designatedreleased drug product −80° C. freezer. The accepted, released drugproduct may be thawed and administered to a patient (e.g., via enema).

Example 5 Example Process for Manufacturing an Oral MRT Composition fromthe Released Drug Product

Step 1—Thaw Suspended Intermediate

Batch manufacturing of an oral MRT composition begins with selection ofmultiple bags of released drug product (e.g., where each bag of releaseddrug product contains a frozen suspended intermediate manufactured asdescribed in Example 4). Each of the bags of released drug product arefrom the same donor. The selected bags are thawed at room temperaturefor at least 2 hours and 500 g of suspended intermediate is transferredinto 1-liter centrifuge bottles.

Step 2—Add Diluent

Phosphate Buffered Saline (PBS) may be added to the SuspendedIntermediate in a 1:1 ratio by weight, mixed by intermittent gentleshaking, and held for 30 minutes at 4° C.

Step 3—Differential Centrifugation

The diluted intermediate may be centrifuged for 2 min at 1,400×g and 4°C.±3° C. The supernatant from this slow speed spin may be transferredinto new 1 L centrifuge bottles containing bottle liners for furtherprocessing, and the pelleted material is discarded. The bottlescontaining the collected supernatant may then centrifuged at 10,000×gfor 45 mins at 4° C.±3° C. After the high-speed spin, the supernatantmay discarded and the recovered microbiota are retained for furtherprocessing.

Step 4—Resuspend the recovered microbiota with lyophilization excipientsolution

A lyophilization excipient/cryoprotectant may be added to the recoveredmicrobiota in a 1:1 ratio (w/w). The lyophilization excipient mayinclude 2.3% polyethylene glycol (e.g., PEG 3350), 10% trehalose, 10%sucrose, and 1% glycerin in purified water. The mixture may be mixedusing a paddle mixer to form a uniform suspension.

Step 5—Lyophilization

The resuspended microbiota solution is aliquoted into 96-well plates(200 μl per well) and placed into a qualified lyophilizer.Lyophilization steps are described in the following table.

TABLE 7 Lyophilization Cycle Details Cycle Parameters Pre-cyclingThermal None Steps Freeze Temp −40 C. Additional Freeze Time 360 minCondenser Set point −40 C. Vacuum Set point 200 mTorr Primary DryingStep Temp Time Vac Ramp/Hold 1 −40 C. 840 min 200 mTorr Hold 1 −35 C.720 min 150 mTorr Hold 3 −30 C. 720 min 150 mTorr Hold 4 −25 C. 640 min100 mTorr Hold 5 −15 C. 360 min 100 mTorr Hold 6  10 C. 240 min 100mTorr Hold 7  20 C. 120 min 100 mTorr Hold Secondary Drying Step TempTime Vac Ramp/Hold I  25 C.  60 min 100 mTorr Hold

Step 6—Milling Operation

Within a NF-grade-nitrogen purged glove box workstation, lyophilizeddrug pellets are milled through a low energy mill.

Step 7—Double Encapsulate

Milled drug is encapsulated in size 0 capsules using manual capsulefilling equipment within an NF-grade nitrogen purged glove box. Contentuniformity among the capsules is tested for the filled capsules. Thesecapsules are then filled into size 00 capsules.

Step 7A—Fill Uniformity Testing (g/capsule)

A representative sample set of filled size 0 capsules is weighed toensure uniformity of fill.

Step 8—Bulk Pack and Label

Capsules are individually enclosed in aluminum-aluminum blisterpackaging with child resistant lidding foil. The blister packs arelabeled and packed in dose cartons, then stored at 5° C.±3° C.

Step 9—Collet Final Drug Product QC, Stability and Reserve Samples

A representative sample of the filled size 00 capsules is tested forproduct release.

Step 9A: Final Drug Product Release Testing

The drug product may be tested for release.

Step 10—Quarantine Hold

Bulk packed capsules are placed in refrigerated storage designated forquarantined drug product until packaging.

Step 11—Final Product Disposition

If all QC test results and batch record review requirements areacceptable, the batch will be dispositioned as accepted. If results arenot acceptable the batch will be dispositioned as rejected anddiscarded. Quality assurance will disposition Final Product batches asaccepted or rejected.

Step 12—Final Pack and Label

Capsules are individually enclosed in aluminum-aluminum blisterpackaging with child resistant lidding foil. The blister packs arelabeled and packed in dose cartons, then stored at 5° C.±3° C.

Step 13—Release

Product batches dispositioned as accepted will be removed fromquarantined drug product storage, labelled “Accepted” and transferred todesignated released drug product storage at 5° C.±3° C.

Processing Hold Times: Hold times for each intermediate step arepresented below in Table 8.

TABLE 8 Processing Hold Times Intermediate Storage Hold step ContainerClosure Conditions Time Resuspended Heat Sealed centrifuge 5° C. ± 3° C.3 days Microbiota bottle liners that Intermediate are heat sealed andplace in centrifuge bottles. LI Pellets Double bagged in 5° C. ± 3° C.30 days poly bags with desiccant between the bags and placed into heatsealed aluminum pouches Filled Size 0 Bagged in poly bags 5° C. ± 3° C.30 days Capsules with desiccant placed into heat sealed aluminum pouchesFilled Size 00 Bagged in poly bags 5° C. ± 3° C. 30 days Capsules withdesiccant and placed into heat sealed aluminum pouches

Example 6 Batch Formula

TABLE 9 Batch Formula of Microbiota Capsule Prior to EncapsulationQuantity per Representative Quality Formulation 9000 g Batch IngredientsReference Function (% w/w) (g/batch) Starting Material Suspended NA Rawmaterial 47.9 9000 Intermediate containing fecal microbiota SuspensionExcipients Phosphate USP Diluent NA 9000 Buffered Saline^(a)Lyophilization excipients Polyethylene NF Cryoprotectant 5.2 23 Glycol3350 Powder Glycerin NF Cryoprotectant 2.2 10 Trehalose NFCryoprotectant 22.4 100 Sucrose NF Cryoprotectant 22.4 100 Purified USPSolvent NA NA Water^(b) NA—not applicable ^(a)Component is removedduring processing; trace amounts of sodium chloride and phosphate saltsmay remain after purification ^(b)Component removed duringlyophilization

The starting material for each batch is 60 released doses of thereleased drug product as described in Example 4, equivalent toapproximately 9000 g starting material, and results in recovery ofapproximately 1700 g of microbiota resuspended in lyophilization medium.This material is lyophilized in 96 well plates is performed whichresults in approximately 7700 lyophilized pellets. After milling of thelyophilized pellets, the powder yield is approximately 300 g. With afill weight of 235 mg for each capsule, the theoretical yield ofcapsules is approximately 1200 capsules/batch. Despite donor to donorvariability in the starting material, the estimated bacteria per mg issimilar among donors and lots manufactured to date. The product iscontrolled by the fill weight.

Example 6 Description and Composition of Drug Product

The drug product is a concentrated, lyophilized microbial powder that isencapsulated in an enteric capsule. The drug product is composed of thefollowing (see Tables 10-12):

TABLE 10 Drug Product Components Quality Ingredients Reference FunctionStarting Material Suspended Intermediate NA Raw material containingfecal microbiota Suspension Excipients Phosphate Buffered Saline USPDiluent Lyophilization excipients Polyethylene Glycol 3350 Powder NFCryoprotectant Glycerin NF Cryoprotectant Trehalose^(b) NFCryoprotectant Sucrose NF Cryoprotectant Purified Water^(c) USP SolventCapsule excipients Titanium Dioxide USP Capsule opacifier Hypromellose2910 USP Capsule structure Hypromellose Acetate Succinate NF Capsulestructure (Hypromellose AS) NA—not applicable ^(a)Component is removedduring processing; trace amounts of sodium chloride and phosphate saltsmay remain after purification ^(b)Referred to as trehalose throughoutthis document but is formally trehalose dihydrate ^(c)Component removedduring lyophilization ^(d)Refer to VCaps ® capsule section 3.2.P.4.1

Milled lyophilized intermediate powder is filled and tamped into size 0capsules without any additional excipients and over-encapsulated withsize 00 capsules. The expected formulation percent concentrations arethe same as the pre-encapsulation material.

TABLE 11 Composition of Lyophilized Intermediate in Each CapsuleFormulation Strength Ingredients (% w/w) (mg/capsule) PurifiedIntermediate 47.9 112.6 Polyethylene Glycol 3350 Powder 5.2 12.2Glycerin 2.2 5.2 Trehalose 22.4 52.6 Sucrose 22.4 52.6 Total 100 235

Table 12 depicts the drug product presentation.

TABLE 12 Drug Product Presentation Capsule Powder Fill Weight CapsuleDosage Form Filled Double- (typical to yield Powder Strength CapsuleEncapsulation Dosage Form Fill (Estimated Size Capsule Size Strength)Tolerance CFU/capsule) 0 00 235 mg ±10% 1 × 10⁷ to 1 × 10¹⁰

It should be understood that this disclosure is, in many respects, onlyillustrative. Changes may be made in details, particularly in matters ofshape, size, and arrangement of steps without exceeding the scope of thedisclosure. The invention's scope is, of course, defined in the languagein which the appended claims are expressed.

What is claimed is:
 1. An oral microbiota restoration therapycomposition, comprising: a capsule; a lyophilized intermediate disposedwithin the capsule, the lyophilized intermediate including fecal-derivedmicrobiota, polyethylene glycol, trehalose, sucrose, and glycerin. 2.The oral microbiota restoration therapy composition of claim 1, whereinthe lyophilized intermediate includes 40-60% (w/w) fecal-derivedmicrobiota.
 3. The oral microbiota restoration therapy composition ofclaim 1, wherein the lyophilized intermediate includes 45-50% (w/w)fecal-derived microbiota.
 4. The oral microbiota restoration therapycomposition of claim 1, wherein the lyophilized intermediate includes1-10% (w/w) polyethylene glycol.
 5. The oral microbiota restorationtherapy composition of claim 1, wherein the lyophilized intermediateincludes 3-8% (w/w) polyethylene glycol.
 6. The oral microbiotarestoration therapy composition of claim 1, wherein the lyophilizedintermediate includes 0.5-5% (w/w) glycerin.
 7. The oral microbiotarestoration therapy composition of claim 1, wherein the lyophilizedintermediate includes 1-4% (w/w) glycerin.
 8. The oral microbiotarestoration therapy composition of claim 1, wherein the lyophilizedintermediate includes 10-40% (w/w) trehalose.
 9. The oral microbiotarestoration therapy composition of claim 1, wherein the lyophilizedintermediate includes 20-30% (w/w) trehalose.
 10. The oral microbiotarestoration therapy composition of claim 1, wherein the lyophilizedintermediate includes 10-40% (w/w) sucrose.
 11. The oral microbiotarestoration therapy composition of claim 1, wherein the lyophilizedintermediate includes 20-30% (w/w) sucrose.
 12. An oral microbiotarestoration therapy composition, comprising: a capsule; a lyophilizedintermediate disposed within the capsule, the lyophilized intermediateincluding fecal-derived microbiota, polyethylene glycol, trehalose, andsucrose.
 13. The oral microbiota restoration therapy composition ofclaim 12, wherein the lyophilized intermediate includes 45-50% (w/w)fecal-derived microbiota.
 14. The oral microbiota restoration therapycomposition of claim 13, wherein the lyophilized intermediate includes3-8% (w/w) polyethylene glycol.
 15. The oral microbiota restorationtherapy composition of claim 14, wherein the lyophilized intermediateincludes 20-30% (w/w) trehalose.
 16. The oral microbiota restorationtherapy composition of claim 15, wherein the lyophilized intermediateincludes 20-30% (w/w) sucrose.
 17. The oral microbiota restorationtherapy composition of claim 16, wherein the lyophilized intermediatefurther comprises glycerin.
 18. The oral microbiota restoration therapycomposition of claim 17, wherein the lyophilized intermediate includes1-4% (w/w) glycerin.
 19. An oral microbiota restoration therapycomposition, comprising: a mechanically milled lyophilized powder, thelyophilized powder including fecal-derived microbiota, polyethyleneglycol, and trehalose.
 20. The oral microbiota restoration therapycomposition of claim 19, wherein the lyophilized powder includes 20-30%(w/w) trehalose.